Phthalocyanine photosensitizers for photodynamic therapy and methods for their use

ABSTRACT

The present invention relates to a series of novel phthalocyanine compositions (or compounds) suitable for use as photosensitizers for photodynamic therapy. Specifically, the invention relates to a series of new aluminum (Al) germanium (Ge), gallium (Ga), tin (Sn) and/or silicon (Si) phthalocyanines having substituted amine or quaternary ammonium axial ligands attached to the central metal, and the use of these new phthalocyanine compositions for the treatment of cancer through photosensitization. Moreover, the present invention is directed to the methods of preparing these compositions for use in photodynamic therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.07/980,494, filed Nov. 23, 1992, now abandoned, which is a continuationapplication of U.S. patent application Ser. No. 554,290, filed Jul. 17,1990, which issued as U.S. Pat. No. 5,166,197, Nov. 24, 1992.

BACKGROUND OF THE INVENTION

The present invention is directed to a series of novel phthalocyaninessuitable for use as photosensitizers for photodynamic therapy. Moreparticularly, the present invention is directed to a series of newaluminum (Al) and silicon (Si) phthalocyanines having substituted amineor quaternary ammonium axial ligands, and the use of these newphthalocyanine compositions for the therapeutic treatment of cancer. Inaddition, the present invention is directed to the methods ofsynthesizing these new compositions.

Photodynamic therapy, hereinafter also referred to as "PDT", is arelatively new process for treating cancer wherein visible light is usedto activate a substance, such as a dye or drug, which then attacks,through one or more photochemical reactions, the tumor tissue therebyproducing a cell killing, or cytotoxic, effect. It has been discoveredthat when certain non-toxic photodynamic sensitizers, such ashematoporphyrin derivative ("HpD" or "Photofrin® I"), which is extractedfrom serum and/or components thereof, are applied intravenously,topically, intradermally, etc., to the human or animal body, they areselectively retained by the cancerous tissue while being eliminated bythe healthy tissue. As a result, after the administration of aphotodynamic substance and the waiting of a certain period of timedepending upon the type of photosensitizer utilized (i.e. two to threedays after HpD treatment), substantially higher levels of thephotosensitizer are retained in the cancerous tissue.

The tumor or cancerous tissue containing the photosensitizer can then beexposed to therapeutic light of an appropriate wavelength and at aspecific intensity for activation. The light can be directly appliedthrough the skin to the cancerous area from a conventional light source(e.g. laser, sun lamp, white light sources with appropriate filters,etc.), or in cases where the cancerous tissue is located deeper withinthe body, through surgical or non-surgical entry such as by the use offiber optic illumination systems, including flexible fiber opticcatheters, endoscopic devices, etc. The light energy and thephotosensitizer cause a photochemical reaction which kills the cell inwhich the photosensitizer resides.

As a result, by applying a photosensitizer to the animal or human body,waiting for a sufficient period of time for the photosensitizer topermeate throughout the body while dissipating from normal tissue morerapidly than from cancer tissue, and exposing the cancerous regionduring the sensitive period to suitable light of sufficient intensity,the preferential destruction of the cancerous tissue will occur.

The mechanisms by which the photosensitizers produce their killingeffect on the host cells upon illumination by an appropriate lightsource are not precisely defined and are the subject of continuingresearch. However, it is thought that there are at least two generalmechanisms by which the photosensitizers are chemically altered uponillumination. The first general reaction mechanism involves energytransfer from the excited photosensitizer to oxygen present in thecancerous tissue. The excited photosensitizer transfers its additionalenergy to the oxygen, producing singlet molecular oxygen (SMO or ¹ O₂)which consequentially alters essential cell components.

More particularly, in the first general reaction mechanism, it isthought that the light energy causes the photosensitizer to becomeexcited from the ground state, S₀, to the first excited singlet state,S₁. The photosensitizer's excited singlet state, S₁, is then transformedby intramolecular coupling to the lowest lying triplet state T₁. Througha direct intermolecular process discussed more particularly by John G.Parker of The John Hopkins University, Baltimore, Md., in U.S. Pat. Nos.4,576,173; 4,592,361; and 4,827,938, the photosensitizer transfers thisenergy to oxygen molecules present in the tissue and raises them fromthe ground triplet to the first excited electronic singlet state ¹ O₂.The singlet molecular oxygen, ¹ O₂, destroys or alters vital cellularcomponents such as the cell membrane, etc., ultimately inducing necrosisand destroying the cancerous tissue.

The process by which biological damage occurs as a result of the opticalexcitation of a photosensitizer in the presence of oxygen is generallyreferred to as "photodynamic action". A more detailed discussionconcerning the use of photodynamic action in the treatment of cancer isdiscussed by Thomas J. Dougherty, William R. Potter, and Kenneth R.Weishaupt of Health Research, Inc., Buffalo, N.Y., in a series ofpatents, i.e. U.S. Pat. Nos. 4,649,151; 4,866,168; 4,889,129; and4,932,934, concerning improved hematoporphyrin and porphyrin derivativesincluding dihematoporphyrin ether (DHE), the purified form of HpD, andmethods utilizing same, for photodynamic therapy.

The second general mechanism thought to be involved in the killingeffect produced by certain photosensitizers involves the production offree radicals. Subsequent reactions of the radicals with organicmolecules and/or with oxygen results in the biochemical destruction ofthe diseased tissue.

Although the exact effective mechanisms of the photochemical reactionswhich produce death of the cancer cells is not clearly understood andvaries depending upon the type of photosensitizer utilized, what isclear is that photodynamic therapy is effective for the preferentialdestruction of cancerous tissue. Furthermore, photodynamic therapy hasseveral attractive features over conventional methods for treatingcancer such as chemotherapy, radiation, surgical procedures, etc., inthat the photosensitizers utilized are generally non-toxic, concentrateor remain preferentially in cancer cells, can be utilized with othermodes of treatment since PDT does not interfere with other chemicals orprocesses, etc.

As a result, photodynamic therapy is now used experimentally for thetreatment of malignant diseases in humans and animals. For example,photodynamic therapy has been used successfully for the treatment of abroad range of cancers including metastatic breast tumors, endometrialcarcinomas, bladder tumors, malignant melanoma, Kaposi's sarcoma, basalcell carcinoma, chondrosarcoma, squamous cell carcinoma, prostatecarcinoma, laryngeal papillomas, mycosis fungoides, superficial cancerof the tracheobronchial tree, cutaneous/mucosal papilloma, gastriccancer, enteric cancer, etc.

The drug in current clinical use is "Photofrin® II" a purified versionof hematoporphyrin derivative (HpD, or "Photofrin® I"). HpD andPhotofrin® II are complex mixtures of substances and have been thesubject of numerous investigations to identify their active compounds.In addition, other porphyrins and porphyrin-like compounds such aschlorins (see U.S. Pat. Nos. 4,656,186; 4,693,885; and 4,861,876) andenlarged porphyrins, naphthalocyanines, phthalocyanines, platyrins,porphycenes (see U.S. Pat. Nos. 4,649,151 and 4,913,907), purpurins,texaphyrins, and verdins have been investigated as photosensitizers.Numerous other substances, such as "merocyanine 540", xanthenes(Rhodamine 123 6 G&B) cationic cyanic dyes, chalcogenapyryllium dyes,phenothiazinium derivatives, tetracycline, berbine sulphate, acridineorange, and fluorescein have also been used as photosensitizers,however, the porphyrin derivatives are generally preferred because theyabsorb in the long wave length region (red region) of the visiblespectrum.

The specific reactions used by many of the above substances to producethe killing effect in cancer cells on exposure to excitatory light arein most instances not known or well understood. As mentioned above,research continues in this area in order to more fully understand thecytotoxic effects produced by the various photosensitizers.

Notwithstanding the above, although many of the above identifiedsubstances have demonstrated enhanced effects in photodynamic therapy,these substances also produce various side effects which limit their usefor photodynamic therapy. The most predominant side effect exhibited bymany of the currently utilized substances is the development ofuncontrolled photosensitivity reactions in patients after the systemicadministration of the photosensitizer and the exposure of the patient tonormal sunlight. In this regard, on exposure to the sun, thephotodynamic therapy patients can develop generalized skinphotosensitization. As a result, the patient after receiving systemicinjections of a photosensitizing substance is required to avoid brightlight, especially sunlight for periods of about four to eight weeks.

Furthermore, since many of the above photosensitizers bind to othernon-cancerous cells, some healthy cell destruction can also occur.Similarly, although many of the photosensitizers are soluble in water,large dosages are required for cellular uptake and/or treatment. Thus,use of many of the above indicated photosensitizers is normally limitedto patients with severe cancerous tumors and continuing research isbeing conducted in order to produce photosensitizing substances, and/ormethods of administering such substances, that avoid these sidereactions as well as produce enhanced photosensitizing effects.

Considerable attention has recently been directed to a group ofcompounds having the phthalocyanine ring system. These compounds, calledphthalocyanines, hereinafter also abbreviated as "Pc", are a group ofphotoactive dyes that are somewhat structurally similar (i.e. havenitrogen containing ring structure) to the porphyrin family.Phthalocyanines are azaporphyrins consisting of four benzoindole nucleiconnected by nitrogen bridges in a 16-membered ring of alternatingcarbon and nitrogen atoms around a central metal atom (i.e. C₃₂ H₁₆ N₈M) which form stable chelates with metal cations. In these compounds,the ring center is occupied by a metal ion (such as a diamagnetic or aparamagnetic ion) that may, depending on the ion, carry one or twosimple ligands. In addition, the ring periphery may be eitherunsubstituted or substituted.

Since E. Ben-Hur and I. Rosenthal disclosed the potential use ofphthalocyanines as photosensitizers in 1985 (E. Ben-Hur and I.Rosenthal, The phthalocyanines: A new class of mammalian cellphotosensitizers with a potential for cancer phototherapy, Int. J.Radiat. Biol. 47, 145-147, 1985), a great deal of research has followedproducing a number of phthalocyanines for photodynamic therapy. Althoughprior studies with phthalocyanines have been generally disappointing,primarily because of the poor solubility characteristics of the basicring, some of these compounds have attractive characteristics.

For example, unlike some of the porphyrin compounds, phthalocyaninesstrongly absorb clinically useful red light with absorption peaksfalling between about 600 and 810 nm (Abernathy, Chad D., Anderson,Robert E., Kooistra, Kimberly L., and Laws, Edward R., Activity ofPhthalocyanine Photosensitizers against Human Glioblastoma in Vitro,Neurosurgery, Vol. 21, No. 4, pp. 468-473, 1987). Although porphyrinsabsorb light poorly in this wavelength region, as a result of theincreased transparency of biological tissues at longer wavelengths, redlight is normally used for photodynamic therapy. Thus, the greaterabsorption of red light by the phthalocyanines over porphyrins indicatesdeeper potential penetration with the phthalocyanines in photodynamictreatment processes.

Furthermore, it has been found that the addition of certain metalcations (i.e. diamagnetic metal cations such as aluminum) to thephthalocyanine ring will, in some instances, create a fairly stablechelate with enhanced photosensitizing tumoricidal activity. While themechanisms for producing the photoreactions are not clear (i.e. it isnot known whether singlet oxygen or hydroxyl radicals, etc. areproduced), the choice of the metal cation is apparently critical in thatcertain metals (i.e., paramagnetic metals) may actually inhibit thephototoxic properties of the resulting compound. Abernathy, et al., pp.470-471.

In addition, the phthalocyanines offer many benefits over the porphyrincomponents as photosensitizers in that the phthalocyanines arerelatively easy to synthesize, purify, and characterize in contrast tothe porphyrins, which are often difficult to prepare. Similarly, themetal phthalocyanines are exceptionally stable compounds in comparisonto the porphyrin or porphyrin-like compounds. As a result, certainmetallic phthalocyanines, such as aluminum phthalocyanine tetrasulfonate(AlPcS) and chloroaluminum phthalocyanine (AlPcCl), offer a number ofadvantages over porphyrins as therapeutic agents for photodynamictherapy.

However, notwithstanding some of the benefits indicated above, only afew of the many possible types of ring-substituted phthalocyaninesbelonging to this group have been examined. By far the most attentionhas been given to sulfonated phthalocyanines and to phthalocyanines withperipheral substituents carrying hydroxy, alkoxy, and aminosubstituents. Very little attention has been given to phthalocyanineswith complex metal ligands.

The limited variety of phthalocyanines which have been tested varygreatly i their photosensitizing activity Metal-free phthalocyaninesshow poor photodynamic activity (Abernathy, C. D., R. E. Anderson, K. L.Kooistra, & E. R. Laws, Jr., "Activity of PhthalocyaninePhotosensitizers Against Human Glioblastoma in vitro", Neurosurgery 21,pp 468-473, 1987; Chan, W. S., J. F. Marshall, G. Y. F. Lam, & I. R.Hart, "Tissue Uptake, Distribution, and Potency of the PhotoactivatableDye Chloroaluminum Sulfonated Phthalocyanine in Mice BearingTransplantable Tumors", Cancer Res.) 48, pp 3040-3044, 1988, Sonoda, M.,C. M. Krishna, & P. Riesz, "The Role of Singlet Oxygen in thePhotohemolysis of Red Blood Cells Sensitized by PhthalocyanineSulfonates", Photochem Photobiol. 46, pp. 625-632, 1987) as dophthalocyanines containing paramagnetic metals. In contrast, thosecontaining diamagnetic metals, such as Al, Sn, and Zn, are active as aresult of the long half-life of the triplet state (Chan, W. S., J. F.Marshall, G. Y. F. Lam, & I. R. Hart, "Tissue Uptake, Distribution, andPotency of the Photoactivatable Dye Chloroaluminum SulfonatedPhthalocyanine in Mice Bearing Transplantable Tumors", Cancer Res. 48,pp. 3040-3044, 1988; Sonoda, M., C. M. Krishna, & P. Riesz, "The Role ofSinglet Oxygen in the Photohemolysis of Red Blood Cells Sensitized byPhthalocyanine Sulfonates", Photochem. Photobiol. 46, pp. 625-632,1987). While in general there appears to be an increase inphotosensitizing ability with lipophilicity (Berg, K., J. C. Bommer, &J. Moan, "Evaluation of Sulfonated Aluminum Phthalocyanines for use inPhotochemotherapy. Cellular Uptake Studies", Cancer Letters 44 pp. 7-15,1989) some highly lipophilic derivatives, such as a tetraneopentoxyderivative, are poor photosensitizers (Rosenthal, I., E. Ben-Hur, S.Greenberg, A. Concepcion-Lam, D. M. Drew, & C. C. Leznoff, "The Effectof Substituents on Phthalocyanine Phototoxicity", Photochem. Photobiol.46, pp. 959-963, 1987).

Recently, Leznoff, et al. (Leznoff, C. C., Vigh, S., Svirskaya, P. I.,Greenberg, S., Drew, D. M., Ben-Hur, E. & Rosenthal, I., "Synthesis andPhotocytoxicity of Some New Substituted Phthalocyanines", Photochem.Photobiol. 49, pp. 279-284, 1989) synthesized a series ofring-substituted phthalocyanines. The substituents were hydroxy oralkoxy groups, as well as substituted amines. Of this series, a Znphthalocyanine with four diethylaminopropyl groups was reported to havesome photosensitizing activity against Chinese hamster fibroblast V79cells in culture. However, it is critical to note that although aminegroups were present in the Zn phthalocyanine compound containing thefour diethylaminopropyl groups, the amine groups were ring substituentsand no simple axial ligands were specified. For some time the applicantshave been searching for phthalocyanines having superior photosensitizingability. In this search, the applicants have emphasized compounds withcomplex metal ligands. Initially, applicants examined thephotocytotoxicity of twenty-one phthalocyanines taken from a collectionin the applicants' laboratories to Chinese hamster fibroblasts, i.e. V79cells. One of these phthalocyanines was HOSiPcOSi(CH₃)₂ (CH₂)₃ OCH₂--CHOHCH₂ N(C₂ H₅).sub. 2, a phthalocyanine composition carrying ahydroxyl amine functional group. This was found to be taken upefficiently by the Chinese hamster fibroblast V79 cells and to haveexcellent photocytotoxicity. However, solutions of this composition indimethylformamide were found to decompose relatively rapidly. Further,it appeared that the composition might have dark toxicity (i.e. be toxicto tissues in the absence of light) in vivo because of its --OCHOHCH₂NR₂ functional group.

With the results of this preliminary work in mind, the applicants thenprepared and studied a series of new aluminum and siliconphthalocyanines having relatively simple ligands carrying NR₂ or NR₃ +functions. The present invention is the result of applicants' studies ofthese compounds, and the use of the same for photodynamic therapy.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a series ofphthalocyanine compounds, (or compositions) with modifying moietieslinked to the central metal, which is either aluminum (Al) germanium(Ge), gallium (Ga), tin (Sn), or silicon (Si). Specifically, the presentinvention relates to a series of aluminum, germanium, gallium, tin orsilicon phthalocyanines having an axial group, or groups, carrying, orterminating in, an amine or quaternary ammonium function. The specificembodiments of the invention can be generally characterized by thefollowing Formula I: ##STR1## wherein M is (G)_(a) Y[(OSi(CH₃)₂(CH₂)_(b) N_(c) (R')_(d) (R")_(e))_(f) X_(g) ]_(p)

wherein:

Y is selected from the group of Si, Al, Ga, Ge, or Sn;

R' is selected from the group of H, C, CH₂, CH₃, C₂ H₅, C₄ H₉, C₄ H₈ NH,C₄ H₈ N, C₄ H₈ NCH₃,, C₄ H₈ S, C₄ H₈ O, C₄ H₈ SE, CH₂ CH₃, (CH₂)₃(CH₃)₂, OC(O)CH₃, OC(O), (CH₃)₂ (CH₂)₁₁, CS, CO, CSE, OH, C₄ H₈ N(CH₂)₃CH₃, (CH₂)₂ N(CH₃)₂, C(O)C₂₇ H₃₀ N₂ O, (CH₂)_(n) N((CH)_(o) (CH₃))₂, analkyl group having from 1 to 12 carbon atoms;

R" is selected from the group of H, SO₂ CH₃, (CH₂)₂ N(CH₃)₂, (CH₂)₁₁CH₃, C(S)NHC₆ H₁₁ O₅, (CH₂)_(n) N((CH)_(o) (CH₃))₂, and an alkyl grouphaving from 1 to 12 carbon atoms;

G is selected from the group of OH, CH₃, and (CH₃)₃ C(CH₃)₂ ;

X is selected from the group of: I; F; Cl; or Br;

a=0 where Y is Al, or 1 where Y is Si;

b=an integer from 2 to 12;

c=0, 1;

d=0, 1, 2, or 3;

e=0, 1, or 2;

f=1 or2;

g=0, 1;

n=an integer from 1 to 12;

o=an integer from 1 to 11;

p=1 or 2;

or preferably, M=

AlOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

AlOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ;

CH₃ SiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NHCSNHC₆ H₁₁ O₅ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ OCOCH₃ ;

Si[OSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ ]_(22I) ⁻ ;

CH₃)₃ C(CH₃)₂ SiOSiOSi(CH₃)₂ (CH₂)₄ NCOC₂₇ H₃₀ N₂ O;

HOSiOSi(CH₃)₂ (CH₂)₃ OH;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O;

AlOSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ I⁻ ;

HOSiOSi(CH₃)₂ (CH₂)₈ N(CH₃)₂ ;

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂)₃ (CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NCS;

HOSiOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ;

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃ CH₃ ; or

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NH]₂ ;

In an additional aspect, the present invention relates to the variousmethods of synthesizing the novel phthalocyanine compositions. The novelphthalocyanines produced by the invention exhibit enhancedcharacteristics which make them well suited for photodynamic therapywhen utilized alone or in combination with a pharmaceutical carrier. Thephthalocyanines of the present invention are also useful asimmunosuppressant and to purge blood of viral components.

In a further aspect, the present invention is directed to variousmethods for destroying cancer tissue comprising the steps ofadministering to the cancer tissue an effective amount of aphthalocyanine composition having an axial group, or groups, carrying,or terminating in an amine or quaternary ammonium function, and applyinglight of sufficient wavelength and intensity to activate the compositionthereby exerting a cell killing, or cytotoxic, effect on the cancertissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings which are presentedfor the purpose of illustrating the invention and not for the purpose oflimiting same.

FIG. 1 is a graph illustrating the photodynamic efficacy of the variouscompositions of the present invention in comparison to AlPcCl. Thephthalocyanine composition compounds of the present invention weretested for their photodynamic efficiency against Chinese hamsterfibroblast V79 cells by colony formation. Monolayer cultures weretreated with the indicated phthalocyanine composition for 18 hours,irradiated with various fluences of red light, and immediatelytrypsinized and replated at appropriate aliquots in triplicate. Coloniesof at least 50 cells were counted after 7-10 days. The platingefficiency of the untreated cells was approximately 90%.

FIG. 2 is a graph demonstrating the percent survival of the compositionsof the present invention in comparison to ALPcCl in relation tointracellular phthalocyanine (nmole/10⁷ cells) and light fluence(kJ/m²). In this regard, in FIG. 2 the data of FIG. 1 were replotted asa function of the product of the amount of cell-associatedphthalocyanine and the light fluence.

FIG. 3 is a graph which compares the percent survival of L5178Y strain Rcells receiving photodynamic therapy and treated with: PcIV, representedby the open circles; PcXII, represented by the solid circles; PcX,represented by the open squares; and PcXVIII, represented by the solidsquares, at varying doses of light.

FIG. 4 shows the tumor volume response of chemically-induced benign skinpapillomas in SENCAR mice, to photodynamic therapy with PcIV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a series of novel phthalocyaninecompositions (or compounds) suitable for use as photosensitizers forphotodynamic therapy. Specifically, the invention relates to a series ofnew aluminum (Al) or Ga and/or silicon (Si) Ge, or Sn phthalocyanineshaving substituted amine or quaternary ammonium axial ligands attachedto the central metal, and the use of these new phthalocyaninecompositions for the treatment of cancer through photosensitization.Moreover, the present invention is directed to the methods of preparingthese compositions for use in photodynamic therapy.

Although research has recently been directed to the use of variousphthalocyanines for photodynamic therapy, this activity has beenprincipally directed to phthalocyanines with peripheral substituents,and little, if any, attention has been given to phthalocyanines withcomplex metal ligands. Along this line, in the phthalocyaninecompositions described in the prior art, only simple ligands, such as Clor OH ligands, are attached to the central metal. However, in the newcompositions of the present invention, axial ligands carrying or,terminating in an amine function or a quaternary ammonium function areattached to the central metal. As a result, it is believed by theapplicants that these more complex axial ligands give the newphthalocyanine compositions the potential to bind to the various speciesthat assist in transporting the composition to and from their targets,as well as enhance the potential for the phthalocyanines to bind totheir specific target cells.

This is demonstrated in that some of the novel phthalocyanines of thepresent invention having substituted amine or quaternary ammonium axialligands attached to either aluminum or silicon as the central metal, aremuch more effective in producing photodynamic activity when comparedwith chloroaluminum phthalocyanine (AlPcCl). The enhanced cytotoxiceffects produced are due to the increased cellular uptake of thecompositions and/or the increased loss of clonogenicity as a functionboth of the concentration of the phthalocyanine and the red lightfluence.

More particularly, in applicants' investigation for phthalocyaninesexhibiting enhanced photosensitizing ability through the synthesis andevaluation of a number of phthalocyanine compositions having complexmetal ligands, the applicants have produced a series of new aluminum andsilicon phthalocyanines having substituted amine or quaternary ammoniumaxial ligands. In this regard, two silicon phthalocyanines and onealuminum phthalocyanine with axial groups terminating in an aminefunction were prepared:

SiPc(CH₃)(OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂),

SiPc(OH)(OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂), and

AlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂.

In addition, two silicon phthalocyanines and one aluminum phthalocyaninewith axial groups terminating in a quaternary ammonium function wereprepared:

SiPc(OH)(OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂)⁺ I⁻,

SiPc(OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃)⁺ I⁻)₂, and

AlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻.

The new phthalocyanine compositions can be generally characterized bythe following formula: ##STR2## wherein M is (G)_(a) Y[(OSi(CH₃)₂(CH₂)_(b) N_(c) (R')_(d) (R")_(e))_(f) X_(g) ]_(p)

wherein:

Y is selected from the group of Si, Al, Ga, Ge, or Sn;

R' is selected from the group of H, C, CH₂, CH₃, C₂ H₅, C₄ H₉, C₄ H₈ NH,C₄ H₈ N, C₄ H₈ NCH₃,, C₄ H₈ S, C₄ H₈ O, C₄ H₈ Se, CH₂ CH₃, (CH₂)₃(CH₃)₂, OC(O)CH₃, OC(O), (CH₃)₂ (CH₂)₁₁, CS, CO, CSe, OH, C₄ H₈ N(CH₂)₃CH₃, (CH₂)₃ N(CH₃)₂, C(O)C₂₇ H₃₀ N₂ O, (CH₂)_(n) N((CH)_(o) (CH₃))₂, analkyl group having from 1 to 12 carbon atoms;

R" is selected from the group of H, SO₂ CH₃, (CH₂)₂ N(CH₃)₂, (CH₂)₁₁CH₃, C(S)NHC₆ H₁₁ O₅, (CH₂)_(n) N((CH)_(o) (CH₃))₂, and an alkyl grouphaving from 1 to 12 carbon atoms;

G is selected from the group of OH, CH₃, and (CH₃)₃ C(CH₃)₂ ;

X is selected from the group of: I; F; Cl; or Br;

a=0 where Y is Al, or 1 where Y is Si;

b=an integer from 2 to 12;

c=0, 1;

d=0, 1, 2, or 3;

e=0, 1, or 2;

f=1 or 2;

g=0, 1;

n=an integer from 1 to 12;

o=an integer from 1 to 11;

p=1 or 2;

or preferably, M=

AlOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

AlOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ;

CH₃ SiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ;

Si[OSi(CH₃)₂ (CH₂)₄ NHCSNHC₆ H₁₁ O₅ ]₂ ;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ OCOCH₃ ;

Si[OSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ ]₂ 2I⁻ ;

(CH₃)₃ C(CH₃)₂ SiOSiOSi(CH₃)₂ (CH₂)₄ NCOC₂₇ H₃₀ N₂ O;

HOSiOSi(CH₃)₂ (CH₂)₃ OH;

Si[OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O;

AlOSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ I⁻ ;

HOSiOSi(CH₃)₂ (CH₂)₈ N(CH₃)₂ ;

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S;

HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂)₃ (CH₃)₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NCS;

HOSiOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ;

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ]₂ ;

HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃ CH₃ ; or

Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NH]₂.

The new phthalocyanine compositions bearing the substituted amine orquaternary ammonium axial ligands have been evaluated for theirphotodynamic efficiency against Chinese hamster fibroblast V79 cells invitro. Chloroaluminum phthalocyanine (AlPcCl) was used as a referencecompound. Along this line, the compounds, SiPc(CH₃)(OSi(CH₃)₂ (CH₂)₃N(CH₃)₂) and SiPc((OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻)₂, displayed lesseffective cellular uptake, and are less preferred. The most efficientphotosensitizer, as judged by uptake, growth delay, andphotocytotoxicity, was SiPc(OH)(OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂. The relatedquaternary ammonium compound, SiPc(OH)OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻),displayed poorer uptake but induced marked photocytotoxicity. Whenexpressed as a function of the

product of intracellular phthalocyanine and the fluence reducing cellsurvival to 10%, this quaternary ammonium compound was the mostefficient photosensitizer.

The specific process utilized to synthesize the aluminum and siliconphthalocyanine compounds of the present invention, and the enhancedresults produced through the use of these new compounds for photodynamictherapy, are more particularly described below in the followingexamples.

EXAMPLES

Synthesis of Phthalocyanines

CH₃ OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ --Under argon gas a solution of CH₃ MgCl intetrahydrofuran (3.0M, 45 mL) was added dropwise to a cool (ice bath)solution of (CH₃ O)₃ Si(CH₂)₃ N(CH₃)₂ (11 mL) in tetrahydrofuran (100mL), and the resulting suspension was stirred for 2 hours while beingkept cool at about 5° C.). Methanol (20 mL) then was added to thesuspension and the mixture formed was filtered. The solid was washedwith ether (50 mL) and the washings and filtrate were combined andconcentrated with a rotary evaporator (45° C.). The concentrate wasfractionally distilled under vacuum (45 torr) and a selected fraction(86°-88° C., 5.0 g.) was retained (55%): NMR (CDCl₃) δ3.42 (s, CH₃ O),2.24 (m, γ-CH₂), 2.20 (s, NCH₃), 1.49 (m, β-CH₂), 0.57 (m, α-CH₂), 0.10(s, CH₃ Si). The compound is a colorless liquid.

AlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ --Compound I. A mixture of CH₃ OSi(CH₃)₂(CH₂)₃ N(CH₃)₂ (203 mg) produced above and a suspension of AlPcOh xH₂ O(56 mg) and 2-ethylpyridine (15 mL) that had been dried by distillation(3 mL of distillate) was refluxed for 45 minutes and filtered. Thefiltrate was evaporated to dryness with a rotary evaporator (˜40° C.)and the solid was dissolved CH₂ Cl₂ (2 mL). Hexanes (3 mL) were added tothe solution and the resulting suspension was filtered. The solid waswashed (benzene and hexanes), vacuum dried (65° C.), and weighed (63 mg,98% assuming AlPcOH 3H₂ O); NMR (C₅ D₅ N, 70° C.) δ9.65 (m, 1,4-PcH),8.28 (m, 2,3-PcH), 1.63 (s, NCH₃), 0.99 (m, γ-CH₂), -0.50 (m, β-CH₂),-1.80 (m, α-CH₂), -2.33 (s, SiCH₃).

The compound is blue and is soluble in CH₂ Cl₂ and toluene.

AlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ --Compound II. A mixture ofAlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ (30 mg), benzene (10 mL), and CH₃ I (15 μL)was refluxed for 1.5 hours, cooled, and filtered. The solid was vacuumdried (60° C.) and weighed (31 mg., 86%): NMR (C₅ D₅ N, 70° C.) δ9.75(m, 1,4-PcH), 8.34 (m, 2,3-PcH), 2.90 (s, NCH₃), 2.02 (m, γ-CH₂), -0.53(m, β-CH₂), -1.87 (m, αCH₂), -2.40 (s, SiCH₃).

The compound is a blue solid and is soluble in CH₂ Cl₂ and CH₃ OH but isinsoluble in toluene and H₂ O.

CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ --Compound III. Procedures in thissynthesis that were carried out under low light conditions (room lightsoff, shades drawn) are identified by the symbol 1. A mixture of CH₃OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ (224 mg) and a suspension of CH₃ SiPcOH (117mg) and pyridine (25 mL) that had been dried by distillation (1) wasslowly distilled (1) for 3 hours (10 mL of distillate) and then filtered(1, no solid). The filtrate was evaporated to dryness with a rotaryevaporator (1, 75° C.), and the solid was dissolved in CH₂ Cl₂ (1, 2mL). Hexanes (30 mL) were added to the solution (1) and the resultingsuspension was filtered (1). The solid was washed (hexanes), vacuumdried (65° C.), and weighed (11 mg, 76%): mp >260° C.; NMR (CDCl.sub. 3)δ9.63 (m, 1,4-PcH), 8.33 (m, 2,3-PcH), 1.74 (s, NCH₃), 1.01 (m, γ-CH₂),-1.18 (m, β-CH₂), -2.25 (m, α-CH₂), -2.96 (s, Si(CH₃)₂), -6.35 (s,SiCH₃).

The compound is dark green and is soluble in CH₂ Cl₂ and toluene.Solutions of it are rapidly photolyzed by white light.

HOSiPcOSi(CH₃)₂ (CH₂)₃ N(₃)₂ --Compound I. A mixture of CH₃SiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ (35 mg), N(C₂ H₅)₃ saturated with H₂ O (0.2mL), and toluene (70 mL) was irradiated with an incandescent light (300W in 35 mm slide projector) for 15 minutes. The resulting suspension wasconcentrated with a rotary evaporator (˜45° C.) and the concentrate (˜5mL) was diluted with hexanes (1 mL). The suspension formed was filteredand the solid was washed (hexanes), vacuum dried (65° C.), and weighed(33 mg, 96%): mp>260° C.; NMR (dimethylformamide-d₇, 70° C.) δ9.68 (m,1,4-PcH), 8.47 (m, 2,3-PcH), 1.52 (s, NCH₃), 0.74 (m, γ-CH₂), -1.11 (m,β-CH₂), -2.27 (m, α-CH.sub. 2), -2.89 (s, SiCH₃). MS-HRFAB exact massm/z calculated for C₃₉ H₃₅ N₉ O₂ Si₂ M+7.17.2452. Found 717.2422.

The compound is blue and is soluble in CH₂ Cl₂ and toluene.

HOSiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ --Compound V. A mixture ofHOSiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ (24 mg), CH₃ I (25 μL), and benzene (10mL) was refluxed for 1.5 hours, cooled, and filtered. The solid waswashed (benzene), vacuum dried (65° C.), and weighed (23 mg, 81%): NMR(dimethylformamide-d₇, 70° C.) δ9.66 (m, 1,4-PcH), 8.45 (m, 2,3-PcH),2.87 (s, NCH₃), 2.06 (m, γ-CH₂), -0.97 (m, β-CH₂), 2.25 (m, α-CH₂),-2.83 (s, SiCH₃). MS-HRFAB exact mass m/z calculated for C₄₀ H₃₈ N₉ O₂Si₂ (M-I)⁺ 732.2687. Found 732.2668.

The compound is blue. It is soluble in CH₂ Cl₂ and CH₃ OH but isinsoluble in toluene and H₂ O.

Sipc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂. A mixture of CH₃ OSi(CH₃)₂ (CH₂)₃N(CH₃)₂ (239 mg) and a suspension of SiPc(OH)₂ (232 mg) and2-ethylpyridine (30 mL) that had been dried by distillation (˜2 mL ofdistillate) was slowly distilled for 2 hours (˜5 mL of distillate). Theresulting solution was filtered, the filtrate was evaporated to drynesswith a rotary evaporator (˜60° C.), and the solid was dissolved in CH₂Cl₂ (3.5 mL). The CH₂ Cl₂ solution was diluted with hexanes (˜40 mL),the suspension formed was filtered, and the solid was washed (hexanes),air dried, and weighed (263 mg, 76%); NMR (CDCl₃), δ9.63 (m, 1,4-PcH),8.34 (m, 2,3-PcH), 1.65 (s, NCH₃), 0.90 (m, γ-CH₂), -1.10 (m, β-CH₂),-2.26 (m, α-CH₂), -2.87 (s, SiCH₃).

The compound is blue and is soluble in CH₂ Cl₂ and toluene.

SiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃)⁺ I⁻ ]₂ --Compound VI. A mixture ofSiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂ produced above (30 mg), CH₃ I (36 μL)and benzene (5 mL) was refluxed for 1.5 hours, cooled, and filtered. Thesolid was washed (benzene, hexanes), vacuum dried (60° C.), and weighed(32 mg, 79%): NMR (CD₃ OD) δ9.63 (m, 1.4-PcH), 8.41 (m, 2,3-PcH), 1.65(s, NCH₃), 0.90 (m, γ-CH₂), -1.10 (m, β-CH₂), -2.21 (m, α-CH₂), -2.90(m, SiCH₃).

The compound is blue and is soluble in CH₂ Cl₂ and CH₃ OH but isinsoluble in toluene. It disperses in H₂ O but doses not dissolve in it.

Additional Phthalocyanine Compounds

SiPc[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂ Compound VII

A mixture of CH₃ OSi(CH₃)₂ (CH₂)₄ NH₂ (100 μL, 0.53 mmol), SiPC(OH)₂ (65mg, 0.11 mmol) and pyridine (15 ml) was distilled for 30 minutes (˜5 mldistillate) and filtered. The filtrate was evaporated to dryness with arotary evaporator (˜70° C.). The solid was dissolved in ethanol (4 ml),precipitated from the solution with water (3 ml), recovered byfiltration, washed (ethanol-water solution, 2:1), vacuum dried (˜60° C.)and weighed (81 mg, 0.097 mmol, 88%): UV-Vis (toluene) λ_(max) 669 nm;NMR (CDCl₃) δ9.67 (m, 1,4-Pc H), 8.36 (m, 2,3-Pc H), 1.71 (t, δ-CH₂),-0.10 (m, γ-CH₂), -1.33 (m, β-CH₂), -2.20 (m, α-CH₂), -2.87 (s, SiCH₃).MS-HRFAB exact mass, m/z: calculated for C₄₄ H₄₈ N₁₀ O₂ Si₃ (M)⁺,832.3270; found, 832.3261, 832.3274. The compound is blue and is solublein CH₂ Cl₂, dimethylformamide, pyridine and ethanol.

HOSiPcOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ Compound X

To prepare CH₃ OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂, a solution ofCH₃ OSi(CH₃)₂ (CH₂)₃ Cl (5.06 g, 30 mmol), CH₃ CH₂ NH(CH₂)₂ N(CH₃)₂ (5.0mL, 61 mmol) and CH₃ OH (5.0 ml) was refluxed for 6 hours and thendistilled under gradually reduced pressure (20 torr final). Theremainder was diluted with ether (20 ml) and filtered. The solid waswashed (ether) and the washings and the filtrate were combined andconcentrated with a rotary evaporator (˜25° C.). The concentrate wasfractionally distilled under vacuum (7 mtorr) and a selected fraction(30°-35° C.) was retained (432 mg, 1.8 mmol, 6%): NMR (CDCl₃) δ3.40 (s,CH₃ O), 2.53 (m, NCH₂ CH₃ and CH₂ CH₂ NCH₃), 2.37 (m, γ-CH₂ and CH₂ CH₂NCH₃), 2.21 (s, NCH₃), 1.46 (m, β-CH₂), 0.97 (t, NCH₂ CH₃), 0.52 (m,α-CH₂), 0.07 (s, SiCH₃). The compound is a colorless oil.

All steps but the finally drying step of this procedure were carried outunder low-intensity illumination. To prepare CH₃ SiPcOSi(CH₃)₂ (CH₂)₃N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂, a mixture of the CH₃ OSi(CH₃)₂ (CH₂)₃ N(CH₂CH₃)(CH₂)₂ N(CH₃)₂ (432 mg, 1.8 mmol) and a suspension of CH₃ SiPcOH(291 mg, 0.51 mmol) and pyridine (120 ml) that had been dried bydistillation (˜23 ml of distillate) was slowly distilled for 3 hours (˜5ml of distillate) and then filtered. The filtrate was evaporated todryness with a rotary evaporator (˜80° C.). The solid was dissolved inCH₂ Cl₂ (1 ml), precipitated from the solution with hexanes (20 ml),recovered by filtration, washed (CH₃ OH and hexanes), vacuum dried (˜90°C.) and weighed (306 mg, 0.39 mmol, 76%): NMR (CD₂ Cl₂) δ6 9.68 (m,1,4-Pc H), 8.40 (m, 2,3-Pc H), 2.01 (s, NCH₃), 1.85 (s, NCH₂ CH₂ N),1.83 (q, NCH₂ CH₃), 0.98 (m, γ-CH₂), 0.61 (t, NCH₂ CH₃), -1.18 (m,β-CH₂), -2.39 (m, α-CH₂), -2.94 (s, Si(CH₃)₂), -6.33 (s, SiPcCH₃). Thecompound is green and is soluble in CH₂ Cl₂ and toluene. Solutions of itare rapidly photolyzed by white light.

To prepare HOSiPcOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂, a mixture ofthe CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ (300 mg, 0.38mmol), toluene (600 ml) and (C₂ H₅)₃ N saturated with H₂ O (2.2 ml) wasirradiated with incandescent light (300 W projector lamp) for 40minutes, and then concentrated with a rotary evaporator (˜70° C.). Theconcentrate (˜5 ml) was diluted with hexanes (2.5 ml) and filtered. Thesolid was washed (toluene), dissolved in CH₂ Cl₂ (2 ml), precipitatedfrom the solution with hexanes (20 ml), recovered by filtration, waswashed (hexanes), vacuum dried (˜90° C.), and weighed (136 mg, 0.17mmol, 45%): UV-vis (toluene) λ_(max) 670 nm; NMR (CD₂ Cl₂, 7.6 mM) δ9.28 (m, 1,4-Pc H), 8.30 (m, 2,3-Pc H), 1.93 (s, NCH₃), 1.77 (s, NCH₂CH₂ N), 1.71 (q, NCH₂ CH₃), 0.85 (m, γ-CH₂), 0.49 (t, NCH₂ CH₃), -1.24(m, β-CH₂), -2.43 (m, α-CH₂), -3.02 (s, SiCH₃). Anal. calculated for C₄₃H₄₄ N₁₀ O₂ Si₂ : C,65.45; H,5.62; N,17.75. Found: C,65.18; H,5.51;N,17.74. The compound is blue. It is soluble in toluene, CH₂ Cl₂,dimethylformamide and ethanol.

SiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂ Compound XII

A mixture of CH₃ OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ (201 mg, 1.1 mmol) and asuspension of SiPc(OH)₂ (232 mg, 0.40 mmol) and 2-ethylpyridine (30 ml)that had been dried by distillation (˜1 ml of distillate) was slowlydistilled for 1.5 hours (˜11 ml of distillate). The resulting solutionwas filtered, and the filtrate was evaporated to dryness with a rotaryevaporator (˜40° C.). The sol id formed was extracted (CH₂ Cl₂ -hexanessolution, 1:4, 15 ml), recovered from the extract by rotary evaporation(˜40° C.), dissolved in CH₂ Cl₂ (1.5 ml), precipitated from the solutionwith hexanes (18 ml), recovered by filtration, washed (hexanes), vacuumdried (˜70° C.) and weighed (110 mg, 0.13 mmol, 33%): UV-vis (toluene)λ_(max) 669 nm; NMR (CDCl₃) δ9.61 (m, 1,4-Pc H), 8.31 (m, 2,3-Pc H),1.55 (s, NCH₃), 0.80 (m, γ-CH₂), -1.14 (m, β-CH₂), -2.29 (m, α-CH₂),-2.89 (s, SiCH₃). MS-HRFAB exact mass, m/z: calculated for C₄₆ H₅₃ N₁₀O₂ Si₃ (M+H)⁺, 861.3661; found, 861.3627, 861.3638. The compound is blueand is soluble in CH₂ Cl₂, dimethylformamide and toluene.

SiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ]₂ Compound XVIII

A mixture of CH₃ OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ (191 mg, 0.77mmol) and a suspension of SiPc(OH)₂ (144 mg, 0.25 mmol) and pyridine (45ml) that had been dried by distillation (˜9 ml of distillate) was slowlydistilled for 1 hours (˜3 ml of distillate) and then filtered. Thefiltrate was evaporated to dryness with a rotary evaporator (˜80° C.),and the solid was extracted (CH₂ Cl₂, 10 ml), recovered from the extractby rotary evaporation (˜40° C.), washed twice (ethanol-water solution,1:4), vacuum dried (˜90° C.) and weighed (123 mg, 0.12 mmol, 48%):UV-vis (toluene) λ_(max) 668 nm; NMR (CDCl₃) δ9.64 (m, 1,4-Pc H), 8.33(m, 2,3-Pc H), 2.03 (s, NCH₃), 1.91 (s, NCH₂ CH₂ N), 1.84 (q, NCH₂ CH₃),1.04 (m, γ-CH₂), 0.64 (t, NCH₂ CH₃), -1.14 (m, γ-CH₂), -2.39 (m, α-CH₂),-2.89 (s, SiCH₃ ). MS-HRFAB exact mass, m/z: calculated for C₅₄ H₇₀ N₁₂O₂ Si₃ (M+H)⁺, 1003.5131; found, 1003.5085, 1003.5100. The compound isblue and is soluble in CH₂ Cl.sub. 2, dimethylformamide and toluene.

HOSiPcOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂ Compound XXVIII

To prepare CH₃ OSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂, a mixture of CH₃OSi(CH₃)₂ (CH₂)₃ Cl (3.05 g, 18 mmol), NH[(CH₂)₃ N(CH₃)₂ ]₂ (8.0 mL, 36mmol), K₂ CO₃ (0.488 g, 3.5 mmol) and CH₃ OH (1.0 ml) was heated in oilbath (˜110° C.) for 48 hours and filtered. The filtrate was fractionallydistilled under vacuum (5 mtorr) and a selected fraction (99°-102° C.),was retained (543 mg): NMR (CDCl₃) δ3.40 (s, CH₃ O), 2.33 (m, CH₂ CH₂CH₂ NCH₃), 2.19 (s, NCH₃), 1.61 (quintet, CH₂ CH₂ CH₂ NCH₃), 1.43 (m,β-CH₂), 0.55 (m, α-CH₂), 0.07 (s, SiCH₃). The product is a yellow oil.

All steps but the finally drying step of this procedure were carried outunder low-intensity illumination . To prepare CH₃ SiPcOSi(CH₃)₂ (CH₂)₃N[(CH₂)₃ N(CH₃)₂ ]₂, a mixture of the crude CH₃ OSi(CH₃)₂ (CH₂)₃N](CH₂)₃ N(CH₃)₂ ]₂ (322 mg) and a suspension of CH₃ SiPcOH (302 mg,0.53 mmol) and pyridine (170 ml) that had been dried by distillation(˜23 ml of distillate) was slowly distilled for 3 hours (˜20 ml ofdistillate) and then filtered. The filtrate was evaporated to drynesswith a rotary evaporator (˜80° C.). The solid was washed (ethanol-watersolution, 1:2) and chromatographed (Al₂ O₃ V, 3.5×15 cm, ethylacetate-CH₃ OH solution, 9:1) and the resulting solid was vacuum dried(˜60° C.) and weighed (194 mg, 0.23 mmol, 43%): NMR (CDCl₃) δ9.60 (m,1,4-Pc H), 8.29 (m, 2,3-Pc H), 2.08 (s, NCH₃), 1.96 (t, CH₂ CH₂ CH₂NCH₃), 1.73 (t, CH₂ CH₂ CH₂ NCH₃), 1.11 (quintet, CH₂ CH₂ CH₂ NCH₃),0.96 (m, γ-CH₂), -1.18 (m, β-CH₂), -2.46 (m, α-CH₂), -2.98 (s,Si(CH₃)₂), -6.39 (s, SiPcCH₃). The compound is green and is soluble inCH₂ Cl₂ and toluene. Solutions of it are rapidly photolyzed by whitelight.

(Pc 27). A mixture of CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂ (180mg, 0.21 mmol), toluene (360 ml), (C₂ H₅)₃ N (18 ml) and H₂ O (1.5 ml)was irradiated with incandescent light (300 W projector lamp) for 25minutes and then evaporated to dryness with a rotary evaporator (˜35°C.). The solid was chromatographed (Al₂ O₃ V, 3×14 cm, ethyl acetate-CH₃OH solution, 9: 1) and the resulting solid was dissolved in CH₂ Cl₂ (2ml), precipitated from the solution with pentane (12 ml ), recovered byfiltration, washed (CH₂ Cl₂ -pentane solution, 1:6; pentane), vacuumdried (˜60° C.) and weighed (74.3 mg, 0.086 mmol, 41%): UV-vis(dimethylformamide) λ_(max) 668 nm; NMR (CD₂ Cl₂, 6.7 mM) δ 9.14 (m,1,4-Pc H), 8.12 (m, 2,3-Pc H), 1.84 (s, NCH₃), 1.71 (t, NCH₂ CH₂ CH₂NCH₃), 1.47 (t, CH₂ CH₂ CH₂ NCH₃), 0.83 (quintet, CH₂ CH₂ CH₂ NCH₃),0.64 (m, γ-CH₂), -1.41 (m, β-CH₂), -2.61 (m, α-CH₂), -3.17 (s, SiCH₃).MS-HRFAB exact mass, m/z: calculated for C₄₇ H₅₃ N₁₁ O₂ Si₂ (M+H)⁺,860.4001; found, 860.4020, 860.4011. The compound is blue and is solublein CH₂ Cl₂, dimethylformamide and toluene.

HOSiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ Compound XXVIII

To prepare CH₃ OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃, a solution of CH₃ OSi(CH₃)₂(CH₂)₃ Cl (3.09 g, 19 mmol), HNC₄ H₈ N(CH₃) (4.0 mL, 36 mmol) and CH₃ OH(1.0 ml) was heated in an oil bath (˜110° C.) for 22 hours and allowedto stand for 8 h. The resultant was decanted and the upper layer wasretained (2.40 g): NMR (CDCl₃) δ3.40 (s, CH₃ O), 2.45 (m, NCH₂ CH₂ N),2.32 (m, γ-CH₂), 2.26 (s, NCH₃), 1.51 (m, β-CH₂), 0.55 (m, α-CH₂), 0.08(s, SiCH₃). The product is a yellow oil.

All steps but the finally drying step of this procedure were carried outunder low-intensity illumination. To prepare CH₃ SiPcOSi(CH₃)₂ (CH₂)₃NC₄ H₈ NCH₃ A mixture of the crude CH₃ OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ (162mg) and a suspension of CH₃ SiPcOH (201 mg, 0.35 mmol) and pyridine (90ml) that had been dried by distillation (˜9 ml of distillate) was slowlydistilled for 3 hours (˜10 ml of distillate) and then filtered. Thefiltrate was evaporated to dryness with a rotary evaporator (˜80° C.).The solid was washed (ethanol-water solution, 1:4), vacuum dried (˜60°C.) and weighed (252 mg, 0.33 mmol, 94%): NMR (7.3 mM, CDCl₃) δ9.61 (m,1,4-Pc H), 8.31 (m, 2,3-Pc H), 2.25 (s, NCH₃), 1.65 (m, NCH₂ CH₂ N),0.90 (m, γ-CH₂), -1.25 (m, β-CH₂), -2.38 (m, α-CH₂), -2.98 (s,Si(CH₃)₂), -6.38 (s, SiPcCH₃). The compound is green and is soluble inCH₂ Cl₂ and toluene. Solutions of it are rapidly photolyzed by whitelight.

A mixture of the CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ (200 mg, 0.26mmol), toluene (400 ml), (C₂ H₅)₃ N (4.0 ml) and H₂ O (1.0 ml) wasirradiated with incandescent light (300 W projector lamp) for 20minutes, and then concentrated with a rotary evaporator (˜70° C.). Theconcentrate (˜5 ml) was diluted with hexanes (3.0 ml) and filtered. Thesolid was washed (toluene), dissolved in CH₂ Cl₂ (6 ml), precipitatedfrom the solution with hexanes (12 ml), recovered by filtration, washed(hexanes), vacuum dried (˜60° C.), and weighed (82.9 mg, 0.11 mmol,42%): UV-vis (dimethylformamide) λmax 668 nm; NMR (CDCl₃, 7.8 mM) δ9.15(m, 1,4-Pc H), 8.18 (m, 2,3-Pc H), 2.16 (s, NCH₃), 1.61 (m, NCH₂ CH₂ N),0.76 (m, γ-CH₂), -1.37 (m, β-CH₂), -2.49 (m, α-CH₂), -3.10 (s, SiCH₃).MS-HRFAB exact mass, m/z: calculated for C₄₂ H₄₀ N₁₀ O₂ Si₂ (M+H)⁺,773.2953; found, 773.2944, 773.2950. The compound is blue and is solublein CH₂ Cl₂, dimethylformamide and toluene.

The following compounds were made in a fashion similar to that used forthe above compounds.

SiPc[OSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ]₂ Compound VIII A solution of CH₃ SO₂Cl, SiPc[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂, (C₂ H₅)₃ N and CH₂ Cl₂ was stirred,and the product was isolated, chromatographed and recrystallized:MS-HRFAB exact mass, m/z: calculated for C₄₆ H₅₂ N₁₀ O₆ S₂ Si₂ (M)⁺,988.2821; found, 988.2817, 988.2777.

HOSiPCOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ Compound IX A mixture of CH₃ OSi(CH₃)₂(CH₂)₄ NH₂, CH₃ SiPcOH and pyridine was partially distilled and theresulting CH₃ SiPcOSi(CH₃)₂ (CH₂)₄ NH₂ was isolated and recrystallized.A solution of this compound, CH₃ SO₂ Cl, (C₂ H₅)₃ N and CH₂ Cl₂ wasstirred and the CH₃ SiPcOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ formed was isolatedand chromatographed. Finally, a mixture of this intermediate, CH₂ Cl₂,H₂ O and (C₂ H₅)₃ N was irradiated with light and the product wasisolated, chromatographed and recrystallized: MS-HRFAB exact mass, m/z:calculated for C₃₉ H₃₅ N₉ O₄ SSi₂ (M)⁺, 781.2071; found, 781.2049,781.2074.

SiPc[OSi(CH₃)₂ (CH₂)₄ NHCSNHC₆ H_(11l) O₅ ]₂ Compound XI A mixture of2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate, SiPc[OSi(CH₃)₂(CH₂)₄ NH₂ ]₂ and benzene was refluxed and the resulting SiPc[OSi(CH₃)₂(CH₂)₄ NHCSNHC₁₄ H₁₉ O₉ ]₂ was isolated. A solution of this compound andCH₃ OH was treated with NH₃ gas and the product was isolated andrecrystallized: MS-HRFAB exact mass, m/z: calculated for C₅₈ H₇₀ N₁₂ O₁₂S₂ Si₃ (M)⁺, 1274.3986; found, 1274.3988, 1274.4024.

HOSiPcOSi(CH₃)₂ (CH₂)₃ OCOCH₃ Compound XIII A mixture of ClSi(CH₃)₂(CH₂)₃ OCOCH₃, CH₃ SiPcOH and pyridine was refluxed, and the resultingCH₃ SiPcOSi(CH₃)₂ (CH₂)₃ OCOCH₃ was isolated A solution of this compoundand toluene was irradiated with light and the product was isolated andrecrystallized: MS-HRFAB exact mass, m/z: calculated for C₃₉ H₃₂ N₈ O₄Si₂ (M)⁺, 732.2085; found, 732.2100, 732.2084

SiPc[OSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ ]₂ 2I⁻ Compound XIV Asolution of CH₃ (CH₂)₁₁ I, SiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ andtetrahydrofuran was refluxed, and the product was isolated andrecrystallized. Anal. calculated for C₇₀ H₁₀₂ I₂ N₁₀ O₂ Si₃ : C,57.84;H,7.07; I,17.46; N,9.64. Found: C,58.19; H,6.52; I,17.40; N,9.04, 9.63,9.63.

(CH₃)₃ C(CH₃)₂ SiOSiPcOSi(CH₃)₂ (CH₂)₄ NCOC₂₇ H₃₀ N₂ O Compound XV Asolution of CH₃ OSi(CH₃)₂ (CH₂)₄ NH₂, (CH₃)₃ C(CH₃)₂ SiOSiPcOH andpyridine was partially distilled and the resulting (CH₃)₃ C(CH₃)₂SiOSiPcOSi(CH₃)₂ (CH₂)₄ NH₂ was isolated A solution of this compound andCH₂ Cl₂ was mixed with a mixture of rhodamine B base, (COCl)₂ andbenzene which had been partially distilled, and the product was isolatedand chromatographed: MS-HRFAB exact mass, m/z: calculated for C₇₂ H₇₅N₁₁ O₄ Si₃ (M)⁺, 1241.5311; found 1241.5295, 1241.5265.

HOSiPCOSi(CH₃)₂ (CH₂)₃ OH Compound XVII A solution of CH₃ SiPcOSi(CH₃)₂(CH₂)₃ OCOCH₃, CH₃ OH, K₂ CO₃ and CH₂ Cl₂ was stirred, the reactionproduct was worked up, and the resulting CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ OH wasisolated. A solution of this compound and toluene was irradiated withlight and the product was isolated and chromatographed: MS-HRFAB exactmass, m/z: calculated for C₃₇ H₃₀ N₈ O₃ Si₂ (M)⁺, 690.1979; found,690.1982, 690.1966.

HOSiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O Compound XIX A solution of CH₃ OSi(CH₃)₂(CH₂)₃ Cl, morpholine and CH₃ OH was refluxed and the resulting CH₃OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O was isolated and distilled. A suspension ofthis compound, CH₃ SiPcOH and pyridine was partially distilled, and theCH₃ SiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O was isolated and recrystallized.Finally, a mixture of this intermediate, toluene, (C₂ H₅)₃ N and H₂ Owas irradiated with light, and the product was isolated andrecrystallized: MS-HRFAB exact mass, m/z: calculated for C₄₁ H₃₇ N₉ O₃Si₂ (M+H)⁺, 760.2636; found, 760.2620, 760.2610.

AlPcOSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂)(CH₂)₁₁ CH₃ I⁻ Compound XXI A mixture ofCH₃ (CH₂)₁₁ I and AlPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ was warmed, and theproduct was isolated and recrystallized: MS-HRFAB exact mass, m/z:calculated for C₅₁ H₅₉ AlIN₉ OSi (M)⁺, 995.3472; found, 995.3444,995.3428

HOSiPcOSi(CH₃)₂ (CH₂)₈ N(CH₃)₂ Compound XXII A solution of CH₂ ═CH(CH₂)₆Br, (CH₃)₂ NNH₂ and ether was stirred, the reaction mixture was workedup with HCl, NaNO₃ and NaOH, and the resulting CH₂ ═CH(CH₂)₆ N(CH₃)₂ wasisolated and distilled. A solution of this compound, (CH₃)₂ SiHCl,CHCl₃, H₂ PtCl₆.xH₂ O and isopropanol was warmed and the CH₃ OSi(CH₃)₂(CH₂)₈ N(CH₃)₂.HCl formed was isolated. Next, a suspension of thisintermediate, CH₃ SiPcOH and pyridine was partially distilled, and theCH₃ SiPcOSi(CH₃)₂ (CH₂)₈ N(CH₃)₂ obtained was isolated andrecrystallized. Finally, a solution of this compound and CH₂ Cl₂ wasirradiated with light and the product was isolated, chromatographed, andrecrystallized: MS-HRFAB exact mass, m/z: calculated for C₄₄ H₄₅ N₉ O₂Si₂ (M+H)⁺, 778.3313; found, 788.3300, 788.3290.

SiPC[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O₂ Compound XXIII A suspension of CH₃OSi(CH₃)₂ (CH₂)₃ NC₄ H_(i) O, SiPc(OH)₂ and pyridine was partiallydistilled, and the product was isolated and recrystallized: MS-HRFABexact mass, m/z: calculated for C₅₀ H₅₆ N₁₀ O₄ Si₃ (M)⁺, 944.3794;found, 944.3750, 944.3780.

HOSiPCOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S Compound XXIV A solution of CH₃OSi(CH₃)₂ (CH₂)₃ Cl, thiomorpholine and CH₃ OH was refluxed and theresulting CH₃ OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S was isolated and distilled. Asuspension of this compound, CH₃ SiPcOH and pyridine was partiallydistilled and the CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S formed was isolatedand recrystallized. Finally, a mixture of this intermediate, toluene,(C₂ H₅)₃ N and H₂ O was irradiated with light, and the product wasisolated, chromatographed and recrystallized: MS-HRFAB exact mass, m/z:calculated for C₄₁ H₃₇ N₉ O₂ SSi₂ (M)⁺, 775.2330; found, 775.2308 7752310.

HOSiPcOSi(CH₃)₂ (CH₂)₃ N(CH₂)₃ CH₃)₂ Compound XXV A solution of CH₃OSi(CH₃)₂ Cl, (CH₃ (CH₂)₃)₂ NH and CH₃ OH was refluxed and the resultingCH₃ OSi(CH₃)₂ (CH₂)₃ N((CH₂)₃ CH₃)₂ was isolated. A suspension of thiscompound, CH₃ SiPcOH and pyridine was partially distilled, and theproduct was isolated and chromatographed. Finally, a mixture of thisintermediate, toluene, (C₂ H₅)₃ N and H₂ O was irradiated with light,and the product was isolated and recrystallized: MS-HRFAB exact mass,m/z: calculated for C₄₅ H₄₇ N₉ O₂ Si₂ (M+H)⁺, 802.3470; found, 802.3434,802.3435

HOSiPcOSi(CH₃)₂ (CH₂)₃ NCS Compound XXVI A mixture of CH₃ OSi(CH₃)₂(CH₂)₃ Cl, KNCS and dimethylformamide was warmed and the resulting CH₃OSi(CH₃)₂ (CH₂)₃ NCS was isolated A mixture of the compound, CH₃ SiPcOHand pyridine was partially distilled and the CH₃ SiPcOSi(CH₃)₂ (CH₂)₃NCS formed was isolated, recrystallized and chromatographed. Finally, asolution of this intermediate and toluene was irradiated with light andthe product was isolated and recrystallized: MS-HRFAB exact mass, m/z:calculated for C₃₈ H₂₉ N₉ O₂ SSi₂ (M)⁺, 731.1704; found, 731.1696,731.1669.

SiPc[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ]₂ Compound XXX A suspension of CH₃OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃, SiPc(OH)₂ and pyridine was partiallydistilled, and the product was isolated and recrystallized: MS-HRFABexact mass, m/z: calculated for C₅₂ H₆₂ N₁₂ O₂ Si₃ (M+H)⁺, 971.4505;found, 971.4460, 971.4489.

HOSiPCOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃ CH₃ Compound XXXI A suspension ofpiperazine, CH₃ (CH₂)₃ Br, toluene and K₂ CO₃ was refluxed, and theresulting HNC₄ H₈ N(CH₂)₃ CH₃ was isolated and distilled. A solution ofthis compound, CH₃ OSi(CH₃)₂ (CH₂)₃ Cl and CH₃ OH was refluxed, and theCH₃ OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃ CH₃ formed was isolated. Next, asuspension of this intermediate, CH₃ SiPcOH and pyridine was partiallydistilled, and the CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃ CH₃ obtainedwas isolated and chromatographed. Finally, a mixture of this compound,toluene (C₂ H₅)₃ N and H₂ O was irradiated with light, and the productwas isolated and recrystallized: MS-HRFAB exact mass, m/z: calculatedfor C₄₅ H₄₆ N₁₀ O₂ Si₂ (M+H)⁺, 815.3422; found, 815.3424, 815.3423.

SiPc[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NH]₂ Compound XXXII A solution of CH₃OSi(CH₃)₂ (CH₂)₃ Cl, piperazine and CH₃ OH was refluxed, and theresulting CH₃ OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NH was distilled. A suspension ofthis compound, SiPc(OH)₂ and pyridine was partially distilled and theproduct was isolated and recrystallized. MS-HRFAB exact mass, m/z:calculated for C₅₀ H₅₈ N₁₂ O₂ Si₃ (M+H)⁺, 943.4192; found, 943.4160,943.4213.

In Vitro Evaluation

Culture of Chinese Hamster V79-379 cells

Chinese hamster V79-379 lung fibroblasts were grown in monolayer culturein McCoy's 5A medium (Gibco Laboratories, Grand Island, N.Y.) augmentedwith 10% calf serum and buffered with 20 mM HEPES (pH 7.4).

Uptake of Phthalocyanines

Total uptake was determined by scraping the phthalocyanine-treatedmonolayer, collecting the cells on a glass-fiber filter, and extractingthe phthalocyanine in ethanol, as previously described by Ramakrishnan,et al., 1989. (Ramakrishnan, N., M. E. Clay, M. F. Horng, A. R. Antunez,& H. H. Evans, "DNA Lesions and DNA Degradation in Mouse Lymphoma L5178YCells After Photodynamic Treatment Sensitized by ChloroaluminumPhthalocyanine", Photochem. Photobiol, in press, 1989). The amount ofdrug was determined by absorption at 674 nm and expressed relative tothe number of cells, as measured in a Coulter cell counter on an aliquotof the cell population. Controls included cells not treated with drug,medium alone, and drug-containing medium without cells. The results ofthe total uptake of the various compositions of the present invention incomparison to AlPcCl are set forth below in Table 1.

Drug Treatment and Light Exposure

The cells were treated with 1 μM AlPcCl (from Eastman Kodak, Rochester,N.Y.) or with phthalocyanine compositions I-VI (0.5-1.0 μM finalconcentration in the medium) for 18 hours by adding the appropriatevolume of a 1.0 mM stock solution in dimethylformamide (DMF) to theculture medium. The growth medium was replaced with 4 ml Hank's balancedsalt solution (HBSS), and the cells were irradiated. The light sourcewas a 500 W tungsten-halogen lamp located approximately 29 inches belowthe surface of a glass exposure tray. The visible light administered tothe cells was filtered to allow passage of only that portion of thevisible spectrum above 600 nm (Lee Primary red filter No. 106, VincentLighting, Cleveland, Ohio). The fluence rate was approximately 0.074kJ/m² /s at the level of the cell monolayer.

Growth Delay

At the time of light exposure, there were approximately 1.5×10⁵ cellsper 25 cm² flask. Following irradiation, the HBSS was replaced by 10 mlof fresh complete growth medium, and the cultures were returned to the37° C. incubator. At various times before and after irradiation,duplicate cultures were trypsinized and counted. Controls includeduntreated cells and cells treated with light alone or drug alone. Inaddition, in each experiment, the drug to be tested was compared to astandard treatment, i.e. 1 μM AlPcCl for 18 hours followed by 12 kJ/m²light. The results of the growth delay analysis for each of thecompositions I-VI in comparison to AlPcCl are set forth in Table 1below.

Clonogenic Cell Survival

Cells were irradiated at a density of approximately 2×10⁶ per 25 cm²flask. Immediately after irradiation, the cell monolayer was treatedwith trypsin, and appropriate aliquots were plated in triplicate to give100 to 200 colonies in each 10-cm Petri dish. Cell survival wasdetermined by the ability of the cells to form colonies containing atleast 50 cells. The response of cells treated with 1 μM AlPcCl and lightwas compared in each experiment.

                                      TABLE 1                                     __________________________________________________________________________    Activities of Several Al and Si Phthalocyanines                               Efficiency Relative to 1 μM (AlPcCl)                                                                       Growth                                                                Conc.   Delay F.sub.10 (AlPcCl)/                                                                   CF.sub.10 (AlPcCl)/              Comp.                                                                             Structure           (μM)                                                                           Uptake                                                                            (12 kJ/m.sup.2)                                                                     F.sub.10 (Pc)                                                                        CF.sub.10 (Pc)                   __________________________________________________________________________        AlPcCl              1.0 1.0 1.0   1.0    1.0                              I   AlPcOSi(CH.sub.3).sub.2)CH.sub.2).sub.3 N(CH.sub.3).sub.2                                         1.0 2.3 2.1   0.94   0.51                             II  AlPcOSI(CH.sub.3).sub.2 (CH.sub.2).sub.3 N(CH.sub.3).sub.3 .sup.+                                 1.0 1.8 3.4   0.99   0.72                             III CH.sub.3 SiPcOSi(CH.sub.3).sub.2 (CH.sub.2).sub.3 N(CH.sub.3).sub.2                               1.0 0.07                                                                              0.05  ND     ND                               IV  HOSiPcOsi(CH.sub.3).sub.2)(CH.sub.2).sub.3 N(CH.sub.3).sub.2                                      0.5 1.3 >3    1.85   3.9                                                      1.0 1.64                                                                              ND    4.25   3.5                              V   HOSiPcOSi(CH.sub.3).sub.2 (CH.sub.2).sub.3 --N(CH.sub.3).sub.3.sup.+          I.sup.-             1.0 0.3 0     0.59   3.0                              VI  SiPc(OSi(CH.sub.3).sub.2 (CH.sub.2 ).sub.3 --N(CH.sub.3).sub.3).sup.+         I.sup.-).sub.2      1.0 0.1 0.05  ND     ND                               __________________________________________________________________________

Results of Testing Compounds I-VI in V79-379 cell culture

All of the compounds have been examined for the extent of cellularuptake after exposure of V79 cells to 1 μM or less in complete medium,and the data of Table 1 are presented relative to the uptake from 1 μMAlPcCl, which was 0.723±0.172 nmole/10⁷ cells (mean ±S. D., 25determinations). Compounds I, II, and IV were taken up into the cellsmore efficiently than was AlPcCl under these conditions. In particular,when the concentration of Compound IV was 1 μM in the medium, the uptakeinto the cells was sufficiently high that some of the uptake andphototoxicity studies were repeated at 0.5 μM. Compounds III, V, and VIwere less effectively incorporated into V79 cells.

Photodynamic action against V79 cells was assessed both by measurementof growth delay and by assay of the loss of clonogenicity. With bothassays, none of the compounds showed any dark toxicity at concentrationsof 1.0 μM or less for up to 18 hours.

The inhibition of V79 culture growth was measured during a three dayperiod following red light irradiation (12 kJ/m²) ofphthalocyanine-pretreated cells. With each of the active compounds, aswell as with AlPcCl, there was an initial decrease in cell density, asdead cells became detached from the monolayer. Thereafter, the cellnumber per flask increased, as living cells grew and divided. The timefor the cell density to recover to the level at the time of lightexposure was considered the growth delay. Cells treated with 1 μM AlPcClfor 18 hours and 12 kJ/m² light were used for comparison purposes ineach experiment and demonstrated a growth delay of approximately 24hours. The ratio of the growth delay for the test photosensitizer andthe growth delay for AlPcCl measured in the same experiment is recordedin Table 1. There was less inhibition of culture growth when cells wereexposed to compounds III, V, or VI as expected from the poor cellularuptake of these drugs. In contrast, substantial inhibition was observedfor compounds I, II, and IV. A value of >3 for compound IV (Table 1)indicates that the cell density had not recovered to the initial levelduring the three day observation period.

Photocytotoxicity of the phthalocyanines compounds I to VI was alsoassessed by clonogenic assay (Table 1, FIG. 1). In all experiments, 1 μMAlPcCl was included for comparison purposes. From the survival curves(FIG. 1), the fluence reducing the cell survival to 10% (F₁₀) wasobtained. The ratio of the F₁₀ for AlPcCl and the F₁₀ for the testcompound is recorded in Table 1. Compounds I and II appear to be nearlyas efficient photosensitizers as AlPcCl, while compound IV (assayed athalf the concentration) was almost twice as efficient as the standardAlPcCl. Clonogenic assays were not conducted for compounds III and VI,since the data on uptake and growth delay suggested that these compoundswould have poor activity. However, in spite of the low efficiency ofcompound V in inhibiting cell growth, survival measurements were madefor this compound, because it was taken up into V79 cells somewhat moreefficiently than compounds III and VI.

In order to take differences in cellular uptake into consideration inthe assessment of the relative efficiency of these phthalocyanines asphotosensitizers of V79 cells, the survival data were replotted againstthe product of intracellular phthalocyanine concentration and lightfluence (FIG. 2). From these curves, the product of intracellularconcentration and light fluence reducing survival to 10% (CF₁₀) wasobtained, and comparisons of the values for AlPcCl and the testcompounds are recorded in Table 1. By this and the other criteria,compound IV appears to be the most efficient photosensitizer. However,when consideration is given to the lesser cell uptake of compound V, itappears to be about as strong a photosensitizer as compound IV.

Discussion of Testing Compounds I-VI in V79 Cell CulturePhotocytotoxicity

The low activity of compounds III and VI appears to be due to poor celluptake. Both of these compounds have functional groups on both faces ofthe phthalocyanine ring, and it is possible that one face of the ringmust be free for proper interaction with target biomolecules. Either Siphthalocyanine with no more than a hydroxyl group on one face (IV) or Alphthalocyanine with one face free of substituents (I and II) allowsefficient cellular uptake as well as a high degree of cellularinactivation. Thus, both tertiary and quaternary amines appear to beefficacious structures. Compound V is an anomaly. Although it hasfeatures on either face of the phthalocyanine ring found on activemolecules, the combination appears not to allow efficient cellularuptake. However, that which is incorporated into the cells has goodphotodynamic activity.

The results of the in vitro biological tests of the new phthalocyaninescompounds I to VI are an important introduction to the design of a newclass of photosensitizers. The results suggest that tertiary andquaternary amines may be an important class of structures to beexplored. The axial ligands of the series of compounds listed in Table 1are simpler than the corresponding ligand of the original diethylaminewhich served as a prototype. The simpler ligands appear to have theadvantages of stability in solution, making them easier to study. Theinstability of the diethylamine precluded precise measurements of theconcentration of the active species at the time of irradiation.Therefore, the true photosensitizing activity of the prototype compoundmay also be high.

Evaluation of Phthalocyanine Compounds VII-XV, XVII-XIX, XXI-XXVIII, andXXX-XXXII Uptake of Phthalocyanine Compounds VII-XV, XVII-XIX,XXI-XXVIII, and XXX-XXXII into V79 Cells

In addition to the phthalocyanine compounds I to VI, several other newphthalocyanine compounds have proven to be effective in treating cancer.V79 cells Chinese hamster lung fibroblasts were cultured using the cellculture methods described above. The phthalocyanines listed in table 2were added to the cultures typically at concentrations of 1 μM, 2 μM,and/or 4 μM and incubated for 18 hours, after which aliquots of thecells were counted and other aliquots were collected on a glass fiberfilter. When the filters were dry, the phthalocyanines were extractedinto ethanol and the absorption determined at the peak wavelength,usually 668 nm. Values were converted to nmoles taken up by 10⁶ cells,using an extinction coefficient of 2.93×10⁵. The cellular uptake of thephthalocyanines are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Uptake of Additional Phthalocyanines Into V79 Cells                                                         n Moles/                                        Pc     n Moles/10.sup.6 cells 10.sup.6                                        Num.   1 μM    2 μM   4 μM cells/μM                               ______________________________________                                        IV     0.7 ± 0.2                                                                             3.1 ± 0.3                                                                            4.6 ± 2.9                                                                          1.1                                       VII     0.2 ± 0.03       1.1 ± 0.5                                                                          0.2                                       VIII    0.1 ± 0.04        0.8 ± 0.01                                                                        0.2                                       IX     0.1 ± 0.1         1.8 ± 0.8                                                                          0.3                                       X      0.6 ± 0.2         3.3 ± 1.4                                                                          0.7                                       XI     0.1                  0.3 ± 0.1                                                                          0.1                                       XII    2.1 ± 1.2         4.6 ± 1.5                                                                          1.6                                       XIII                        1.7 ± 0.3                                                                          0.4                                       XIV    0.03 ± 0.01       0.05 ± 0.01                                                                        <0.05                                     XV     0.01 ± 0.01       0.14 ± 0.12                                                                        <0.05                                     XVI    0.2 ± 0.2          0.7 ± 0.20                                                                        0.2                                       XVII                        1.7 ± 0.2                                                                          0.4                                       XVIII  0.3 ± 0.1         3.6 ± 0.6                                                                          0.3*                                      XIX    0.3 ± 0.1         2.4 ± 0.5                                                                          0.3*                                      XXI    1.2 ± 0.2         5.8 ± 0.4                                                                          1.3                                       XXII                                ND                                        XXIII                               ND                                        XXIV   0.003 ± 0.001     1.3 ± 0.1                                                                          <0.05*                                    XXV    0.02 ± 0.02       1.5 ± 0.3                                                                          <0.05*                                    XXVI                                ND                                        XXVII  1.8                   5.0 ± 0.01                                                                        1.5                                       XXVIII 1.2 ± 0.2                                                                             3.6 ± 1.0                                                                            11.4 ± 0.05                                                                        1.2*                                      XXX                                 ND                                        XXXI              0.61 ± 0.1     0.3                                       ______________________________________                                         In the last column, wherever possible, a composite value was calculated,      in order to have a single number for the purposes of ranking the uptake       efficiency of the compounds. For most compounds, the average of all the       data has been calculated and rounded to the first decimal. Where all          values are <0.05, the data are presented as <0.05. An asterisk (*)            indicates that an average uptake value, which is the average of the           phthalocyanine doses would be higher than the listed value which is for 1     μM.                                                                   

It appears from Table 2 that the uptake of PcXVIII, PcXIX, PcXXIV,PCXXV, and PcXXVIII are not linearly dependent upon the phthalocyanineconcentration in the medium. PcIV, PcXII, PcXXI, PcXXVII and PcXXVIIIare taken up particulary well by the V79 cells.

Clonogenicity studies using Phthalocyanine Compounds VII-XV, XVII-XIX,XXI-XXVIII, and XXX-XXXII into V79 Cells

Using the cell culture methods described above, V79 cells Chinesehamster lung fibroblasts were treated with either 0.5 or 1.0 μM of thephthalocyanines listed in Table 3. About 18 hours thereafter, the cellswere irradiated with increasing doses of 675 nm broad band red lightfrom a 500 W tungsten-halogen lamp fitted with a 600 nm high passfilter, to determine the light dosage that would kill 90% of thephthalocyanine treated cells. Where 90% of the cells were not killed,the maximum percent of cells killed were determined, (expressed as %survival) and the related light dosage recorded. The results arepresented in Table 3.

                  TABLE 3                                                         ______________________________________                                        EVALUATION OF PHTHALOCYANINE COMPOUNDS                                        IN KILLING V79 CELLS                                                          USING PHOTODYNAMIC THERAPY                                                                                        n                                                                    Maximum  Moles/10.sup.6                                                       Effect   cells/μM                                       Concn.  LD 90      (% survival                                                                            (from                                     Pc      (μM) (kJ/m.sup.2)                                                                             at kJ/m.sup.2)                                                                         Table 2)                                  ______________________________________                                        IV      0.5     4                   1.1                                       VII#    0.5     4                   0.2                                       VIII    1                  94% at 30                                                                              0.2                                       IX      0.5                44% at 9 0.3                                       X       0.5     7                   0.7                                       XI      1                  100% at 20                                                                             0.1                                       XII     0.5     3.3                 1.6                                       XIII    1                  88% at 15                                                                              0.4                                       XIV     1                  93% at 10                                                                              <0.05                                     XV      4                  81% at 20                                                                              0.05                                      XVI     4                  100% at 10                                                                             0.2                                       XVII    1                  19% at 10                                                                              0.4                                       XVIII   1       7                   0.3*                                      XIX     1                  81% at 10                                                                              1.3                                       XXI     0.5     15*                 ND                                        XXII    0.5     10                  ND                                        XXIV    0.5                100% at 10                                                                             <0.05                                     XXV     0.5                87% at 8 <0.05                                     XXVI    1                  100% at 30                                                                             ND                                        XXVII   0.5     6.8                 1.5                                       XXVIII  0.5     1.8                 1.2*                                      XXX*                       30% at 10                                                                              ND                                        XXXI    0.5                30% at 10                                                                              0.3                                       ______________________________________                                         *not totally soluble at 0.5 mM                                                #Preplated data only                                                     

As shown in Table 3, PcIV, PcVII, PcXII, and PcXXVIII achieved the LD 90at the lowest light dosage, and thus are the most activephotsensitizers, that is they are the most active at killing V79 cells.

For comparison, the phthalocyanine uptake values presented in Table 2were also presented in the last column of Table 3. As shown in Table 3,some, but not all, of the differences in photosensitizing activity amongphthalocyanines can be explained by differences in uptake. For example,PcXXVIII which has the highest activity in killing V79 cells of all ofthe phthalocyanines also has a high uptake. The uptake of Pc XXVIII at 1μM is less than that for PcXII, whereas its photodynamic killingefficiency is superior to PcXII when analyzed at 0.5 μM.

It is not surprising that often phthalocyanines with poor uptake arerelatively less active in photodynamic therapy, whereas the most activephthalocyanines demonstrate a relatively high uptake. However, uptakeand activity are not always correlated. For example, PcVII has pooruptake but one of the better photosensitizers. PcXIX has poor uptake butis less active as a photosensitizer, whereas PcXVIII, with similaruptake, demonstrated good activity. Many factors contribute todetermination of the photosensitizer efficiency, including physicalstate in the cells and localization.

Assessment of Photodynamic Efficiency of Additional Phthalocyanines inL5178Y-R Cells

Mouse lymphoma L5178y-R (hereinafter also referred to as "LY-R") cellswere grown in suspension culture as described in Ramakrishnan N.,Oleinick, N. L. Clay, M. E., Horng, M. F., Antunez, A. R., and Evans H.H., DNA lesions and DNA degradation in mouse lymphoma L5178Y cells afterphotodynamic treatment sensitized by chloroaluminum phthalocyanine.Photochem. Photobiol. 50, 373-378, 1989 and Agarwal, M. L., Clay, M. E.,Harvey, E. J., Evans, H. H., Antunez, A. R., and Oleinick, N. L.Photodynamic therapy induces rapid cell death by apoptosis in L5178Ymouse lymphoma cells. Cancer Res., 51, 5993-5996, 1991.

The cells were used while in exponential growth. Stock solutions ofeither 0.5 or 1 mM of PcIV, PcXII, PcX, PcXVIII were prepared indimethylformamide unless otherwise indicated and added to the 10 mLmedium at a rate of 1 μL per mL. After allowing 18 hours for uptake ofthe phthalocyanine into the cells, the flasks containing the cultureswere placed on a glass exposure tray above a 500-W tungsten-halogenlamp. The exposure tray was fitted with a 600-nm high-pass filter.Flasks were exposed to various fluences of red light (up to 30 kJ/m²) ata fluence rate of approximately 74 W/m²). After irradiation, the cellswere collected by centrifugation.

For measurement of clonogenic cell survival, aliquots were plated inmedium containing soft agar as described in Ramakrishnan N., Oleinick,N. L. Clay, M. E., Horng, M. F., Antunez, A. R., and Evans H. H., DNAlesions and DNA degradation in mouse lymphoma L5178Y cells afterphotodynamic treatment sensitized by chloroaluminum phthalocyanine.Photochem. Photobiol. 50, 373-378, 1989. The aliquots were plated insufficient numbers to produce 50-200 colonies. The dishes were kept inan incubator at 37° C. in an atmosphere of 5% CO₂ and 95% air for 10-14days to allow viable cells to form colonies. Colonies were counted byeye. Controls treated with the phthalocyanine alone had platingefficiencies of ˜90%. The plating efficiencies of the treated cells arenormalized to the plating efficiencies of control cells in eachexperiment. For measurement of the induction of apoptosis, DNA wasisolated from the treated and control cells 2 hours after photodynamictherapy, subjected to electrophoresis on 1.5% agarose, stained withethidium bromide, and visualized by UV transillumination, as describedin Agarwal et. al. The results are shown in Tables 4, 5 and 6 and inFIG. 3.

                                      TABLE 4                                     __________________________________________________________________________    Comparison of Different Phthalocyanine Compounds                              In PDT-treated LY-R cells                                                     LIGHT                                                                         DOSE Pc IV   Pc XII    Pc X       Pc XVIII                                    (kJ/m.sup.2                                                                        AVG.                                                                              SD  AVG. SD   AVG.  SD   AVG.                                                                              SD                                      __________________________________________________________________________    0    100     100       100        100                                         1    80.9                                                                              11.4                                                                              82.2 8.6                                                         2    19.7                                                                              2.9 5.23 0.86 71.8  15.4 81.8                                                                              6.0                                     2.5  0.82                                                                              0.09                                                                              0.90 0.15                                                        3    0.16                                                                              0.10                                                                              0.15 0.01 30.1  3.7  73.6                                                                              4.8                                     4            0.014                                                                              0.002                                                                              20.5  1.1  64.0                                                                              7.0                                     5    0.014                                                                             0.001                                                                             0.0027                                                                             0.0008                                                                             0.43  0.19 52.1                                                                              6.2                                     6                      0.031 0.014                                                                              33.8                                                                              5.8                                     8                      0.00058                                                                             0.0003                                                                             9.13                                                                              1.52                                    10                                3.0 3.0                                     __________________________________________________________________________

In Table 4 each phthalocyanine was present at 0.5 μM, and the normalizedplating efficiencies are presented as mean and standard deviation ateach fluence tested. The results show that all four phthalocyanines areactive photosensitizers for photodynamic therapy. Based on theirrelative ability upon irradiation with various fluences of red light toreduce tumor cell survival, these phthalocyanines are ranked from themost active photosensitizers to the least active: PcIV, PcXII, PcX,PcXVIII. This relative activity of these four phthalocyanines is thesame as obtained from screening in V79 cells.

FIG. 3 shows the average plating efficiencies from Table 4 plottedagainst the fluence for each Pc.

                  TABLE 5                                                         ______________________________________                                        Clonogenic Assay of Phthalocyanines                                                   Concentration                                                         Pc      (μM)      LD.sub.50 (kJ/m.sup.2)                                                                   LD(.sub.90 (kJ/m.sup.2)                       ______________________________________                                        Pc IV   0.5 μM    1.38       2.15                                          Pc X    0.5 μM    2.38       4.19                                          Pc XII  0.5 μM    1.11       1.70                                          Pc XVIII                                                                              0.5 μM    5.00       7.81                                          ______________________________________                                    

Table 5 shows the fluence that reduces the cell survival to 50% and to10% and which are given as LD₅₀ and LD₉₀, respectively. The most activecompound of the phthalocyanines shown in Table 5 is PcXII. PcXII whenpresent in the culture medium at 0.5 μM requires less light, that is thelowest fluence, to kill either 50% or of the cells. PcIV is about 80% asactive as PcXII, PcX is 44% as active as PcXII and PcXVIII is 22% asactive as PcXII.

                  TABLE 6                                                         ______________________________________                                        Relative Capacity of Phthalocyanines to Induce Apoptosis                               Minimum Demonstrated Condition                                                  Concentration                                                                              Fluence  C × F                                  Pc         (μM)      (kJ/m.sup.2)                                                                           (μm × kJ/m.sup.2)                   ______________________________________                                        Pc IV      0.4          3.0      1.2                                          Pc VII     0.5          3.0      1.5                                          Pc IX      0.3          12.0     3.6                                                     0.5          8.0      4.0                                                     1.0          12.0     12.0                                         Pc X       0.5          6.0      3.0                                                     1.0          3.0      3.0                                          Pc XII     0.4          3.0      1.2                                          Pc XVIII   0.5          10.0     5.0                                                     1.0          3.0      3.0                                          Pc XXI     0.5          15.0     7.5                                          Pc XXII    0.5          10.0     5.0                                          Pc XXVIII  0.3          3.0      0.9                                          Pc XXX     0.5          15.0     7.5                                          (DMF-Tween 80)                                                                Pc XXXII   0.5          5        2.5                                          (DMF-Tween 80)                                                                ______________________________________                                    

Table 6 shows that photodynamic therapy with the phthalocyaninecompounds listed causes L5178Y cells to undergo apoptosis as the mode ofcell death. Cells were treated with various concentrations of thecompounds listed in the table and various light fluences. DNA gels wereprepared and examined for the characteristic "ladder" pattern of DNAfragments. For each Pc, the minimum total PDT dose tested (calculated asthe product of the minimum phthalocyanine concentration and the minimumfluence) which produced visible DNA fragments is recorded. PcXXX andPcXXXII were not soluble in DMF and were suspended and partiallysolubilized in DMF/Tween 80 for this assay. PcIX is unusual in that itsactivity increases and then decreases as the concentration is raised.PcX was added at concentrations of 0.5 and 1.0 μM; the same minimumvalue for the C×F product was obtained in both cases. PcXVIII was alsoadded at 0.5 and 1.0 μM. The minimum value of C×F differed only slightlyfor the two conditions. PcV, PcVI, PcVIII, PcXI, PcXIV and PcXV, whenevaluated at a concentration of 1 μ M at a fluence of 30 kJ/m² did notinduce apoptosis. Compound PcXVI at a concentration of 4 μM and afluence of 20kJ/m² for 2 hours did not induce apoptosis.

In vivo Evaluation of Phthalocyanine Compounds VII-XV, XVII-XIX,XXI-XXVII, and XXX-XXXII

The relative effectiveness at reducing tumor volume of selectedphthalocyanine compounds at a given dosage was compared in vivo. RIF-1,i.e., radiation-induced fibrosarcoma, tumors were implanted into thebacks of C3H/HeN mice. One tumor was implanted per mouse. Each of thephthalocyanine compounds listed in Table 7 was sonicated and vortexed incorn oil to produce a suspension. When the tumors reached 5-7 cm indiameter and 2-3 mm in thickness, each mouse received 1 mg/kg in 0.1 mlof the corn oil, of the phthalocyanine suspension. For comparison,select mice received a conventional photosensitizer; either 5 mg/kg ofchloroaluminum phthalocyanine tetrasulfonate, herein also referred to as"AlPcTS" in phosphate buffered saline or 5 mg/kg of Photofrin®-II in 5%dextrose. Twenty-four hours after the photosensitizers wereadministered, the tumors were irradiated with visible radiationdelivered by an argon-pumped dye laser. The mice that received aphthalocyanine photosensitizer received light having a wavelength of 675nm and the mice that received the Photofrin® II photosensitizer receivedlight having a wavelength of 630 nm. Each tumor received 135 J/cm² ofradiation. Tumor size was measured every day using calipers. The initialtumor volume was 50±10 mm³. Tumor volume was calculated according to thehemiellipsoid model by the formula: ##EQU1## Where l is length Where Wis width

Where H is height

The tumor response is shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Comparative Responses of RIF-1 Implanted Tumors to                            PDT With Select Phthalocyanine Compounds                                                  Tumor       Doubling Time of Initial                                          Responses   Tumor Volume after PDT                                Photosensitizer                                                                           at 24 hours in days                                               ______________________________________                                        Pc XXVIII   complete    24                                                    Pc XII      complete    20                                                    Pc IV       near complete                                                                             16                                                    Pc XVIII    near complete                                                                             12                                                    Pc IX       near complete                                                                             11                                                    Pc V        moderate     6                                                    Pc VIII     slight       4                                                    AlPcTS*     substantial  7                                                    II*tofrin ™                                                                            near complete                                                                             12                                                    controls                 4                                                    ______________________________________                                         complete- no evidence of any tumor mass in any animal; only the scar from     the photodynamic therapy was evident.                                         near completeno evidence of any tumor mass in four or five animals; only      some tumor mass in one or two animals.                                        substantial a significant tumor shrinkage occurred in all animals. In som     animals the tumor response was complete, yet in others the response was       not complete.                                                                 moderate some tumor shrinkage was evident in some animals. In animals wit     some tumor shrinkage, scar formation was evident.                             slightsome tumor decrease occurred in one or two mice.                   

While the tumor volume in the control mice doubled in four days, thedoubling of tumor volume was delayed in the animals treated with each ofthe compounds except PcVIII. PcXXVIII, PcXII, PcIV, PcXVIII, PcIX wereparticularly effective in reducing tumor volume.

Histological examination of tumors treated with PcIV revealed thepresence of apoptotic bodies in the tumor. Analysis of tumors treatedwith Pc IV showed DNA fragments whose sizes were multiples of 180-200base pairs.

As can be seen from Table 7, Pc XXVIII, Pc XII and Pc IV significantlyimpair the growth of the tumors and are the most preferredphotosensitizers for the treatment of cancer, because of effectivenessat set dosage of phthalocyanine.

Not only do the phthalocyanine compounds of the present invention reducetumor volume, they are capable of eliminating tumors completelyparticularly upon multiple exposures to radiation.

Complete inhibition of tumors by PDT with PcIV

As occurs with PF-II-PDT, regrowth of tumors from the tumor marginsoccurred in the animals treated Pc IV, followed by the exposure tolight. This regrowth possibly originates from the cells which somehowescape irradiation.

To overcome the regrowth, RIF-1 tumors were implanted in C3H/HeN mice,and the mice were treated with PcIV followed by multiple exposures tolight. For multiple exposures to light to be successful, the tumortissue must retain sufficient levels of the photosensitizer over theexposure period.

Since pharmacokinetic data indicated that Pc IV is retained in tumortissue even after 7 days of its administration, Pc IV was administeredonce at the dose of 1 mg/kg body weight in corn oil or entrapped in DPPCliposomes. Thereafter, the tumors were irradiated with an argon ionpumped dye laser tuned at 675 nm for the total light dose of 135 J/cm²(75 mW/cm²). The tumors were irradiated with multiple exposures of 675nm laser light, at varying times, as shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Responses of RIF-1 implanted tumors tO PcIV followed by                       multiple exposures to light                                                            % of Mice Surviving                                                  day of     corn oil    liposomes                                                                              liposomes                                     exposure   15 days     30 days  120 days                                      ______________________________________                                        2          100         100      N/A                                           2 and 3    100         100      N/A                                           2, 3, and 4                                                                              100          0       0                                             2, 3, 4, 5 and                                                                           100          0       0                                             2-6        100          0       0                                             2 and 7    100         100      N/A                                           ______________________________________                                    

Where Pc IV was given in corn oil, regrowth of tumors was evident 15days after photodynamic therapy in all the multiple exposure protocols.However, when the PcIV was administered entrapped in DPPC liposomes,complete tumor cure was evident in those mice which were irradiatedthree, four or five times at an interval of 24 hours. No tumor regrowthoccurred even at 120 days after the photodynamic therapy. Indeed, at thetime the mice were sacrificed 300 days after the light treatment, therewas no evidence of tumor regrowth. Tumor regrowth occurred 30 days afterphotodynamic therapy only in those animals which were irradiated onlyone or two times either at 24 or 120 hour intervals. One reason for thisdifferential effect may be related to the pharmacokinetics of the dye,that is the dye may have been retained in the tissue for a long periodwhich permitted multiple exposures to be effective. Alternatively, theadministration of Pc IV, via DPPC liposomes may enhance uptake andretention of PcIV by the tumor cells.

Squamous Cell Carcinoma

A single cell suspension of human squamous cell carcinoma was injectedsubcutaneously into the back of Harlen-Sprague Dawley athymic nude mice.Thereafter on day 15 the mice were injected with 5 mg/kg of Pc IVsuspended in 0.1 ml corn oil For comparison 5 mg/kg body weight ofPhotofrin® was administered. The results are shown below in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    Tumor Response and Cure following Photodynamic Therapy                                  675 nm                                                                   Pc IV                                                                              Light                                                                              675 nm                                                                              Illumi-                                                  No of                                                                              Concen-                                                                            Dose Power nation                                                   Test tration                                                                            Density                                                                            Density                                                                             Time                                                                              % Tumor                                                                             % Tumor                                        Animals                                                                            (mg/kg)                                                                            (J/cm.sup.2)                                                                       (nW/cm.sup.2)                                                                       (min)                                                                             Response.sup.a                                                                      Cure.sup.b                                     __________________________________________________________________________    5    0.0  75   75    15   0    0                                              5    1.0   0    0     0   0    0                                              5    1.0  35   75    15  40    0                                              5    1.0  75   75    15  80    60                                             5    1.0  135  75    15  100   100                                            __________________________________________________________________________     .sup.a Tumor flat, necrotic, measured 24 hours post illumination.             .sup.b No tumor at 7 days post treatment.                                

As can be seen from Table 9, 1 mg/Kg Pc IV followed by 135 J/cm² of 675nm light at a power Density of 75 mW/cm² for 15 minutes eliminated thetumors in 100% of the mice.

Treatment of chemically induced skin tumors.

6-week-old female SENCAR mice received a single topical application of 5μg DMBA in 0.2 ml acetone on the dorsal skin as tumor initiator. Oneweek later, the animals were started on twice-weekly topicalapplications of 1 μg TPA in 0.2 ml acetone as tumor promoter. All of theanimals developed tumors at 12 weeks. Mice that developed 4-5 tumors peranimal averaging 5-8 mm in diameter and 2-5 mm in thickness were used.Pc IV, entrapped in DPPC liposomes was administered intraperitoneally atdoses of either 0.5 or 1.0 mg/kg and 24 hrs later the tumor area wasilluminated with light from an argon pumped dye laser tuned at 675 nmfor a total light dose of 135 J/cm² (75 mW/cm²). All possible controlswere included; either the animals were untreated, treated only withlaser light or treated only with Pc IV alone.

Curves for animals after PDT with Pc IV at the doses of 0.5 and 1.0mg/kg are shown by d and e in Figure 4. As shown in FIG. 4 the micetreated with PcIV and light showed a decrease in tumor volume whicheventually decreased to 0 volume, that is, no tumor was measurable. Thetumor did not return for the length of the study, 34 days. In contrast,the control tumor volume consistently increased over time.

The invention has been described with reference to the preferredembodiment. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

In addition, although the present invention has been described withreference to the effectiveness of the phthalocyanine compositions inphotodynamic therapy for the destruction of cancer tissue, it is wellunderstood by those skilled in the art that the compositions of theinvention may be well suited for other therapeutic purposes. Along thisline, it is contemplated that other possible uses of the composition ofthe present invention include:

(1) the purging of bone marrow for autologous bone marrowtransplantation;

(2) the purging of viruses from whole blood or blood components;

(3) the treatment of psoriasis;

(4) the treatment of warts;

(5) the treatment of macular degeneration; and

(6) the treatment of intra-arterial plaques.

Thus, the new phthalocyanine compositions of the present invention maybe effective for a wide variety of therapeutic uses.

Dr. E. Ben-Hur and his assciates at the New York blood Center, New YorkN.Y., have demonstrated 11 that compounds V and VI, XIV, and XXI areeffective at purging viruses from blood and/or blood components. Inaddition, the phthalocyanines are useful for study and research ofphotodynamic therapy particularly photodynamic therapy for cancer.

We claim:
 1. A phthalocyanine compound having the following formula:##STR3## wherein M is (G)_(a) Y [(OSi(CH₃)₂ (CH₂)_(b) N_(c) (R')_(d)(R")_(e))_(f) X_(g) ]_(p) wherein:Y is selected from the groupconsisting of Si, Al, Ga, Ge, and Sn; R' is selected from the groupconsisting of H, CH₂, CH₃, C₂ H₅, C₄ H₉, C₄ H₈ NH, C₄ H₈ NCH₃, C₄ H₈ S,C₄ H₈ O, C₄ H₈ Se, CH₂ CH₃, (CH₂)₃ (CH₃), OC(O)CH₃, CS, CO, CSe, OH, C₄H₈ N(CH₂)₃ CH₃, (CH₂)₃ N(CH₃)₂, C(O)C₂₇ H₃₀ N₂ O, (CH₂)_(n) N((CH₂)_(o)(CH₃))₂, and an alkyl group having from 1 to 12 carbon atoms; R" isselected from the group consisting of H, SO₂ CH₃, (CH₂)₂ N(CH₃)₂,(CH₂)₁₁ CH₃, C(S)NHC₆ H₁₁ O₅, (CH₂)_(n) N((CH₂)_(o) (CH₃))₂, and analkyl group having from 1 to 12 carbon atoms; G is selected from thegroup consisting of OH, CH₃, and (CH₃)₃ C(CH₃)₂ SiO; X is selected fromthe group consisting of: I; F; Cl; and Br; a=0 where Y is Al or Ga, or 1where Y is Si, Ge, or Sn; b=an integer from 2 to 12; c=0 or 1; d=0, 1, 2or 3; e=0, 1, or 2; f=1 or 2; g=0 or 1; n=an integer from 1 to 12; o=aninteger from 1 to 11; and p=1 or 2; where M is not AlOSi(CH₃)₂ (CH₂)₃N(CH₃)₂ ; AlOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ; CH₃ SiOSi(CH₃)₂ (CH₂)₃N(CH₃)₂ ; HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ; HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺I⁻ ; Si[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ]₂ ; or Si[OSi(CH₃)₂ (CH₂)₃N(CH₃)₂ ]₂.
 2. The phthalocyanine compound of claim 1, whereinM=Si[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂ ; Si[OSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ]₂ ;HOSiOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ; HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂N(CH₃)₂ ; Si[OSi(CH₃)₂ (CH₂)₄ NHCSNHC₆ H₁₁ O₅ ]₂ ; HOSiOSi(CH₃)₂ (CH₂)₃OCOCH₃ ; Si[OSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ ]₂ 2I⁻ ; (CH₃)₃C(CH₃)₂ SiOSiOSi(CH₃)₂ (CH₂)₄ NCOC₂₇ H₃₀ N₂ O; HOSiOSi(CH₃)₂ (CH₂)₃ OH;Si[OSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂ ]₂ ; HOSiOSi(CH₃)₂ (CH₂)₃NC₄ H₈ O; AlOSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ I⁻ ; HOSiOSi(CH₃)₂(CH₂)₈ N(CH₃)₂ ; Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O]₂ ; HOSiOSi(CH₃)₂ (CH₂)₃NC₄ H₈ S; HOSiOSi(CH₃)₂ (CH₂)₃ N((CH₂)₃ CH₃)₂ ; HOSiOSi(CH₃)₂ (CH₂)₃NCS; HOSiOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂ ; HOSiOSi(CH₃)₂ (CH₂)₃ NC₄H₈ NCH₃ ; Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ]₂ ; HOSiOSi(CH₃)₂ (CH₂)₃ NC₄H₈ N(CH₂)₃ CH₃ ; or Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NH]₂.
 3. The compound ofclaim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₄ NH₂ ]₂.
 4. The compound ofclaim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃ ]₂.
 5. The compoundof claim 2 wherein M is HOSiOSi(CH₃)₂ (CH₂)₄ NHSO₂ CH₃.
 6. The compoundof claim 2 wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ N(CH₂ CH₃)(CH₂)₂ N(CH₃)₂.7. The compound of claim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₄ NHCSNHC₆ H₁₁O₅ ]₂.
 8. A phthalocyanine compound having the following formulaSiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₂ ]₂.
 9. The compound of claim 2 wherein Mis HOSiOSi(CH₃)₂ (CH₂)₃ OCOCH₃.
 10. The compound of claim 2 wherein M isSi[OSi(CH₃)₂ (CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ ]₂ 2I⁻.
 11. The compound ofclaim 2 wherein M is (CH₃)₃ C(CH₃)₂ SiOSiOSi(CH₃)₂ (CH₂)₄ NCOC₂₇ H₃₀ N₂O.
 12. The compound of claim 2 wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ OH. 13.The compound of claim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₃ N(C₂ H₅)(CH₂)₂N(CH₃)₂ ]₂.
 14. The compound of claim 2 wherein M is HOSiOSi(CH₃)₂(CH₂)₃ NC₄ H₈ O.
 15. The compound of claim 2 wherein M AlOSi(CH₃)₂(CH₂)₃ N⁺ (CH₃)₂ (CH₂)₁₁ CH₃ I⁻.
 16. The compound of claim 2 wherein Mis HOSiOSi(CH₃)₂ (CH₂)₈ N(CH₃)₂.
 17. The compound of claim 2 wherein Mis Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ O]₂.
 18. The compound of claim 2 wherein Mis HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ S.
 19. The compound of claim 2 wherein Mis HOSiOSi(CH₃)₂ (CH₂)₃ N((CH₂)₃ CH₃)₂.
 20. The compound of claim 2wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ NCS.
 21. The compound of claim 2wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ N[(CH₂)₃ N(CH₃)₂ ]₂.
 22. The compoundof claim 2 wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃.
 23. Thecompound of claim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈ NCH₃ ]₂. 24.The compound of claim 2 wherein M is HOSiOSi(CH₃)₂ (CH₂)₃ NC₄ H₈ N(CH₂)₃CH₃.
 25. The compound of claim 2 wherein M is Si[OSi(CH₃)₂ (CH₂)₃ NC₄ H₈NH]₂.
 26. A therapeutic composition comprising the phthalocyanine ofclaim 1 and a pharmaceutical carrier therefor.
 27. A method for treatingfibrosarcomas squamous cell carcinoma, and skin tumors comprising thesteps of administering, to a patient an effective amount of thephthalocyanine of claim 1, and applying light of sufficient wave lengthand intensity to the fibrosarcoma, squamous cell carcinoma or skin tumorto activate said phthalocyanine, wherein said activated phthalocyanineexerts a cyotoxic effect on said fibrosarcoma, squamous cell carcinomaor skin tumor.
 28. The method of claim 27, wherein said light is of thevisible spectrum above about 600 nm.
 29. The method of claim 27, whereinthe M group of said phthalocyanine is HOSiOSi(CH₃)₂ (CH₂)₄ NHSO₃ CH₃.30. A method for treating fibrosarcomas, squamous cell carcinoma andskin tumors comprising the steps of administering an effective amount ofa phthalocyanine wherein the phthalocyanine is HOSiPcOSi(CH₃)₂ (CH₂)₃N(CH₃)₂, and applying light of sufficient wave length and intensity tothe fibrosarcoma, squamous cell carcinoma or skin tumor to activate saidphthalocyanine, wherein said activated phthalocyanine exerts a cytotoxiceffect on said fibrosarcoma, squamous cell carcinoma or skin tumor. 31.A method for treating fibrosarcomas, squamous cell carcinoma, and skintumors comprising the steps of administering, to a patient, an effectiveamount of a phthalocyanine, and applying light of sufficient wave lengthand intensity tot he fibrosarcoma, squamous cell carcinoma or skin tumorto activate said phthalocyanine, wherein said activated phthalocyanineexerts a cytotoxic effect on said fibrosarcoma, squamous cell carcinomaor skin tumor andwherein said phathalocyanine is SiPc[OSi(CH₃)₂ (CH₂)₃N(CH₃)₂ ]₂.
 32. The method of claim 27, wherein the M group of saidphthalocyanine is Si[OSi(CH₃)₂ (CH₂)₃ N(C₂ H₅)(CH₂)₂ N(CH₃)₂ ]₂.
 33. Themethod of claim 27, wherein said phthalocyanine is HOSiPcOSi(CH₃)₂(CH₂)₃ NC₄ H₈ NCH₃.
 34. A phthalocyanine compound having the followingformula: CH₃ SiPcOSi(CH₃)₂ (CH₂)₃ N(CH₃)₂.
 35. A phthalocyanine compoundhaving the following formula:SiPc[OSi(CH₃)₂ (CH₂)₃ N(CH₃)₃ ⁺ I⁻ ]₂.